Four-color dna sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

ABSTRACT

This invention provides a process for sequencing single-stranded DNA by employing a nanopore and modified nucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/988,321, filed May 24, 2018, which is a continuation of U.S.application Ser. No. 15/354,531, filed Nov. 17, 2016, now U.S. Pat. No.10,000,801, issued Jun. 19, 2018, which is a continuation of U.S.application Ser. No. 14/242,487, filed Apr. 1, 2014, now U.S. Pat. No.9,528,151, issued Dec. 27, 2016, which is a continuation of U.S.application Ser. No. 13/665,588, filed Oct. 31, 2012, now abandoned,which is a continuation of U.S. application Ser. No. 13/023,283, filedFeb. 8, 2011, now U.S. Pat. No. 8,298,792, issued Oct. 30, 2012, whichis a continuation of U.S. application Ser. No. 12/312,903, filed Jul. 9,2009, now U.S. Pat. No. 7,883,869, issued Feb. 8, 2011, which is a § 371national stage of PCT International Application No. PCT/US2007/024646,filed Nov. 30, 2007, and claims the benefit of U.S. ProvisionalApplication No. 60/872,240, filed Dec. 1, 2006, the contents of each ofwhich are hereby incorporated by reference in their entireties into thisapplication.

Throughout this application, various publications are referenced inparentheses by number. Full citations for these references may be foundat the end of the specification immediately preceding the claims. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

This invention was made with government support under grant numberP50-HG002806 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

DNA sequencing is driving genomics research and discovery. Thecompletion of the Human Genome Project has set the stage for screeninggenetic mutations to identify disease genes on a genome-wide scale (1).Accurate high-throughput DNA sequencing methods are needed to explorethe complete human genome sequence for applications in clinical medicineand health care. To overcome the limitations of the currentelectrophoresis-based sequencing technology (2-5), a variety of newDNA-sequencing methods have been investigated. Such approaches includesequencing by hybridization (6), mass spectrometry based sequencing(7-9), sequence-specific detection of single-stranded DNA usingengineered nanopores (10) and sequencing by ligation (11). Morerecently, DNA sequencing by synthesis (SBS) approaches such aspyrosequencing (12), sequencing of single DNA molecules (13) andpolymerase colonies (14) have been widely explored.

The concept of DNA sequencing by synthesis (SBS) was revealed in 1988with an attempt to sequence DNA by detecting the pyrophosphate groupthat is generated when a nucleotide is incorporated in a DNA polymerasereaction (15). Pyrosequencing which was developed based on this conceptand an enzymatic cascade has been explored for genome sequencing (16).However, there are inherent difficulties in this method for determiningthe number of incorporated nucleotides in homopolymeric regions of thetemplate. Additionally, each of the four nucleotides needs to be addedand detected separately, which increases the overall detection time. Theaccumulation of un-degraded nucleotides and other components could alsolower the accuracy of the method when sequencing a long DNA template. Itis thus desirable to have a simple method to directly detect a reportergroup attached to the nucleotide that is incorporated into a growing DNAstrand in the polymerase reaction rather than relying on a complexenzymatic cascade. The SBS scheme based on fluorescence detectioncoupled with a chip format has the potential to markedly increase thethroughput of DNA sequencing projects. Consequently, several groups haveinvestigated such a system with an aim to construct an ultrahigh-throughput DNA sequencing method (17-18). Thus far, no completesuccess of using such a system to unambiguously sequence DNA has beenpublished.

Previous work in the literature exploring the SBS method is mostlyfocused on designing and synthesizing a cleavable chemical moiety thatis linked to a fluorescent dye to cap the 3′-OH group of the nucleotides(19-21). The rationale is that after the fluorophore is removed, the3′-OH would be regenerated to allow subsequent nucleotide addition.However, no success has been reported for the incorporation of such anucleotide with a cleavable fluorescent dye on the 3′ position by DNApolymerase into a growing DNA strand. The reason is that the 3′ positionon the deoxyribose is very close to the amino acid residues in theactive site of the polymerase, and the polymerase is therefore sensitiveto modification in this area of the ribose ring, especially with a largefluorophore (22).

SUMMARY OF THE INVENTION

This invention provides a method for determining the sequence of a DNAcomprising performing the following steps for each residue of the DNA tobe sequenced:

-   -   (a) contacting the DNA with a DNA polymerase in the presence        of (i) a primer and (ii) four nucleotide analogues under        conditions permitting the DNA polymerase to catalyze DNA        synthesis, wherein (1) the nucleotide analogues consist of an        analogue of dGTP, an analogue of dCTP, an analogue of dTTP or        dUTP, and an analogue of dATP, (2) each nucleotide analogue        comprises (i) a base selected from the group consisting of        adenine, guanine, cytosine, thymine or uracil, and analogues        thereof, (ii) a deoxyribose, (iii) a moiety cleavably linked to        the 3′-oxygen of the deoxyribose and (iv) a unique label        cleavably linked to the base, so that a nucleotide analogue        complementary to the residue being sequenced is incorporated        into the DNA by the DNA polymerase, and (3) each of the four        analogues has a unique label which is different than the unique        labels of the other three analogues;    -   (b) removing unbound nucleotide analogues;    -   (c) again contacting the DNA with a DNA polymerase in the        presence of (i) a primer and (ii) four reversible terminators        under conditions permitting the DNA polymerase to catalyze DNA        synthesis, wherein (1) the reversible terminators consist of an        analogue of dGTP, an analogue of dCTP, an analogue of dTTP or        dUTP, and an analogue of dATP, (2) each nucleotide analogue        comprises (i) a base selected from the group consisting of        adenine, guanine, cytosine, thymine or uracil, and analogues        thereof, which base does not have a unique label bound        thereto, (ii) a deoxyribose, and (iii) a moiety cleavably linked        to the 3′-oxygen of the deoxyribose;    -   (d) removing unbound reversible terminators;    -   (e) determining the identity of the nucleotide analogue        incorporated in step (a) via determining the identity of the        corresponding unique label, with the proviso that step (e) can        either precede step (c) or follow step (d); and    -   (f) following step (e), except with respect to the final DNA        residue to be sequenced, cleaving from the incorporated        nucleotide analogues the unique label, if applicable, and the        moiety linked to the 3′-oxygen atom of the deoxyribose,    -   thereby determining the sequence of the DNA.

This invention also provides a kit for performing the method of claim 1,comprising, in separate compartments,

-   -   (a) nucleotide analogues of (i) GTP, (ii) ATP, (iii) CTP        and (iv) TTP or UTP, wherein each analogue comprises (i) a base        selected from the group consisting of adenine, guanine,        cytosine, thymine or uracil, or an analogue thereof, (ii) a        deoxyribose, (iii) a cleavable moiety bound to the 3′-oxygen of        the deoxyribose and (iv) a unique label bound to the base via a        cleavable linker,    -   (b) reversible terminators comprising a nucleotide analogue        of (i) GTP, (ii) ATP, (iii) CTP and (iv) TTP or UTP, wherein        each analogue comprises (i) a base selected from the group        consisting of adenine, guanine, cytosine, thymine or uracil, or        an analogue thereof, which base does not have a unique label        bound thereto, (ii) a deoxyribose, and (iii) a cleavable moiety        bound to the 3′-oxygen of the deoxyribose;    -   (c) reagents suitable for use in DNA polymerization; and    -   (d) instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A chip is constructed with immobilized DNA templates that areable to self-prime for initiating the polymerase reaction. Fournucleotide analogues are designed such that each is labeled with aunique fluorescent dye on the specific location of the base through acleavable linker, and a small chemically reversible moiety (R) to capthe 3′-OH group. Upon adding the four nucleotide analogues and DNApolymerase, only the nucleotide analogue complementary to the nextnucleotide on the template is incorporated by polymerase on each spot ofthe chip (step 1). A 4 color fluorescence imager is used to image thesurface of the chip, and the unique fluorescence emission from thespecific dye on the nucleotide analogues on each spot of the chip willyield the identity of the nucleotide (step 2). After imaging, the smallamount of unreacted 3′-OH group on the self-primed template moiety iscapped by excess ddNTPs and DNA polymerase to avoid interference withthe next round of synthesis or by 3′-O-allyl-dNTPs to synchronize theincorporation (step 3). The dye moiety and the R protecting group willbe removed to generate a free 3′-OH group with high yield (step 4). Theself-primed DNA moiety on the chip at this stage is ready for the nextcycle of the reaction to identify the next nucleotide sequence of thetemplate DNA (step 5).

FIG. 2. Structures of 3′-O-allyl-dCTP-allyl-Bodipy-FL-510(λ_(abs (max))=502 nm; λ_(em (max))=510 nm), 3′-O-allyl-dUTP-allyl-R6G(λ_(abs (max))=525 nm; λ_(em (max))=550 nm), 3′-O-allyl-dATP-allyl-ROX(λ_(abs (max))=585 nm; λ_(em (max))=602 nm), and3′-O-allyl-dGTP-allyl-Bodipy-650 (λ_(abs (max))=630 nm; λ_(em (max))=650nm).

FIG. 3. The polymerase extension scheme (left) and MALDI-TOF MS spectraof the four consecutive extension products and their deallylatedproducts (right). Primer extended with 3′-O-allyl-dUTP-allyl-R6G (1),and its deallylated product 2; Product 2 extended with3′-O-allyl-dGTP-allyl-Bodipy-650 (3), and its deallylated product 4;Product 4 extended with 3′-O-allyl-dATP-allyl-ROX (5), and itsdeallylated product 6; Product 6 extended with3′-O-allyl-dCTP-allyl-Bodipy-FL-510 (7), and its deallylated product 8.After 30 seconds of incubation with the palladium/TPPTS cocktail at 70°C., deallylation is complete with both the fluorophores and the3′-O-allyl groups cleaved from the extended DNA products.

FIG. 4. DNA extension reaction performed in solution phase tocharacterize the four different chemically cleavable fluorescentnucleotide analogues (3′-O-allyl-dUTP-allyl-R6G,3′-O-allyl-dGTP-allyl-Bodipy-650, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dCTP-allyl-Bodipy-FL-510). After each extension reaction, theDNA extension product is purified by HPLC for MALDI-TOF MS measurementto verify that it is the correct extension product. Pd-catalyzeddeallylation reaction is performed to produce a DNA product that is usedas a primer for the next DNA extension reaction.

FIG. 5. Preparation of azide-functionalized glass chip through a PEGlinker for the immobilization of alkyne labeled self-priming DNAtemplate for SBS.

FIG. 6. (A) Reaction scheme of SBS on a chip using four chemicallycleavable fluorescent nucleotides. (B) The scanned 4-color fluorescenceimages for each step of SBS on a chip: (1) incorporation of3′-O-allyl-dGTP-allyl-Cy5; (2) cleavage of allyl-Cy5 and 3′-allyl group;(3) incorporation of 3′-O-allyl-dATP-allyl-ROX; (4) cleavage ofallyl-ROX and 3′-allyl group; (5) incorporation of3′-O-allyl-dUTP-allyl-R6G; (6) cleavage of allyl-R6G and 3′-allyl group;(7) incorporation of 3′-O-allyl-dCTP-allyl-Bodipy-FL-510; (8) cleavageof allyl-Bodipy-FL-510 and 3′-allyl group; images (9) to (25) aresimilarly produced. (C) A plot (4-color sequencing data) of rawfluorescence emission intensity at the four designated emissionwavelength of the four chemically cleavable fluorescent nucleotides.

FIG. 7. Structures of 3′-O-allyl-dATP, 3′-O-allyl-dCTP, 3′-O-allyl-dGTP,and 3′-O-allyl-dTTP.

FIG. 8. (A) 4-color DNA sequencing raw data with our sequencing bysynthesis chemistry using a template containing two homopolymericregions. The individual base (A, T, C, G), the 10 repeated A's and the 5repeated A's are clearly identified. The small groups of peaks betweenthe identified bases are fluorescent background from the DNA chip, whichdoes not build up as the cycle continues. (B) The pyrosequencing data ofthe same DNA template containing the homopolymeric regions (10 T's and 5T's). The first 4 individual bases are clearly identified. The twohomopolymeric regions (10 A's) and (5 A's) produce two large peaks,making it very difficult to determine the exact sequence from the data.

FIG. 9. Single base extension reaction and MALDI-TOF MS of3′-O-Allyl-dUTP-allyl-R6G.

FIG. 10. Single base extension reaction and MALDI-TOF MS of3′-O-Allyl-dATP-allyl-ROX (39).

FIG. 11. Single base extension reaction and MALDI-TOF MS of3′-O-Allyl-dGTP-allyl-Bodipy-650 (43) and 3′-O-allyl-dGTP-allyl-Cy5(44).

FIG. 12. Single base extension reaction and MALDI-TOF MS of3′-O-Allyl-dCTP-allyl-Bodipy-FL-510 (23).

DETAILED DESCRIPTION OF THE INVENTION Terms

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

A—Adenine; C—Cytosine;

DNA—Deoxyribonucleic acid;

G—Guanine;

RNA—Ribonucleic acid;

T—Thymine; and U—Uracil.

“Nucleic acid” shall mean any nucleic acid molecule, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

“Type” of nucleotide refers to A, G, C, T or U.

“Mass tag” shall mean a molecular entity of a predetermined size whichis capable of being attached by a cleavable bond to another entity.

“Solid substrate” shall mean any suitable medium present in the solidphase to which an antibody or an agent may be affixed.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Embodiments of the Invention

This invention provides a method for determining the sequence of a DNAcomprising performing the following steps for each residue of the DNA tobe sequenced:

-   -   (a) contacting the DNA with a DNA polymerase in the presence        of (i) a primer and (ii) four nucleotide analogues under        conditions permitting the DNA polymerase to catalyze DNA        synthesis, wherein (1) the nucleotide analogues consist of an        analogue of dGTP, an analogue of dCTP, an analogue of dTTP or        dUTP, and an analogue of dATP, (2) each nucleotide analogue        comprises (i) a base selected from the group consisting of        adenine, guanine, cytosine, thymine or uracil, and analogues        thereof, (ii) a deoxyribose, (iii) a moiety cleavably linked to        the 3′-oxygen of the deoxyribose and (iv) a unique label        cleavably linked to the base, so that a nucleotide analogue        complementary to the residue being sequenced is incorporated        into the DNA by the DNA polymerase, and (3) each of the four        analogues has a unique label which is different than the unique        labels of the other three analogues;    -   (b) removing unbound nucleotide analogues;    -   (c) again contacting the DNA with a DNA polymerase in the        presence of (i) a primer and (ii) four reversible terminators        under conditions permitting the DNA polymerase to catalyze DNA        synthesis, wherein (1) the reversible terminators consist of an        analogue of dGTP, an analogue of dCTP, an analogue of dTTP or        dUTP, and an analogue of dATP, (2) each nucleotide analogue        comprises (i) a base selected from the group consisting of        adenine, guanine, cytosine, thymine or uracil, and analogues        thereof, which base does not have a unique label bound        thereto, (ii) a deoxyribose, and (iii) a moiety cleavably linked        to the 3′-oxygen of the deoxyribose;    -   (d) removing unbound reversible terminators;    -   (e) determining the identity of the nucleotide analogue        incorporated in step (a) via determining the identity of the        corresponding unique label, with the proviso that step (e) can        either precede step (c) or follow step (d); and    -   (f) following step (e), except with respect to the final DNA        residue to be sequenced, cleaving from the incorporated        nucleotide analogues the unique label, if applicable, and the        moiety linked to the 3′-oxygen atom of the deoxyribose,    -   thereby determining the sequence of the DNA.

This invention also provides the instant method, wherein step (e) isperformed before step (c).

This invention also provides the instant method, wherein the moietycleavably linked to the 3′-oxygen of the deoxyribose is chemicallycleavable or photocleavable. This invention also provides the instantmethod, wherein the moiety cleavably linked to the 3′-oxygen of thedeoxyribose in the nucleotide analogs of step (a) is an allyl moiety ora 2-nitrobenzyl moiety.

This invention also provides the instant method, wherein the moietycleavably linked to the 3′-oxygen of the deoxyribose in the reversibleterminators of step (c) is an allyl moiety or a 2-nitrobenzyl moiety.

This invention also provides the instant method, wherein the uniquelabel is bound to the base via a chemically cleavable or photocleavablelinker.

This invention also provides the instant method, wherein the uniquelabel bound to the base via a cleavable linker is a dye, a fluorophore,a chromophore, a combinatorial fluorescence energy transfer tag, a masstag, or an electrophore.

This invention also provides the instant method, wherein the moiety ischemically cleavable with Na₂PdCl₄/P(PhSO₃Na)₃. This invention alsoprovides the instant method, wherein the linker is chemically cleavablewith Na₂PdCl₄/P(PhSO₃Na)₃.

This invention also provides the instant method, wherein the primer is aself-priming moiety.

This invention also provides the instant method, wherein the DNA isbound to a solid substrate. This invention also provides the instantmethod, wherein the DNA is bound to the solid substrate via 1,3-dipolarazide-alkyne cycloaddition chemistry. This invention also provides theinstant method, wherein the DNA is bound to the solid substrate via apolyethylene glycol molecule. This invention also provides the instantmethod, wherein the DNA is alkyne-labeled. This invention also providesthe instant method, wherein the DNA is bound to the solid substrate viaa polyethylene glycol molecule and the solid substrate isazide-functionalized. This invention also provides the instant method,wherein the DNA is immobilized on the solid substrate via an azidolinkage, an alkynyl linkage, or biotin-streptavidin interaction.

This invention also provides the instant method, wherein the solidsubstrate is in the form of a chip, a bead, a well, a capillary tube, aslide, a wafer, a filter, a fiber, a porous media, or a column. Thisinvention also provides the instant method, wherein the solid substrateis gold, quartz, silica, plastic, glass, diamond, silver, metal, orpolypropylene. This invention also provides the instant method, whereinthe solid substrate is porous.

This invention also provides the instant method, wherein about 1000 orfewer copies of the DNA are bound to the solid substrate. This inventionalso provides the instant invention wherein 1×10⁷, 1×10′ or 1×10⁴ orfewer copies of the DNA are bound to the solid substrate.

This invention also provides the instant method, wherein the fournucleotide analogues in step (a) are 3′-O-allyl-dGTP-allyl-Cy5,3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dUTP-allyl-R6G. This invention also provides the instantmethod, wherein the four nucleotide analogues in step (a) are3′-O-allyl-dGTP-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-allyl-Bodipy-650,3′-O-allyl-dATP-allyl-ROX and 3′-O-allyl-dUTP-allyl-R6G. This inventionalso provides the instant method, wherein the four nucleotide analoguesin step (a) are 3′-O-allyl-dGTP-allyl-Bodipy-650,3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dUTP-allyl-R6G.

It is understood that in other embodiments the nucleotide analogues arephotocleavable. For example, 2-nitrobenzyl can replace any of the allylmoieties in the analogues described herein. For example,3′-O-2-nitrobenzyl-dGTP-allyl-Bodipy-650,3′-O-2-nitrobenzyl-dGTP-2-nitrobenzyl-Bodipy-650,3′-O-allly-dGTP-2-nitrobenzyl-Bodipy-650. One of skill in the art wouldrecognize various other chemically cleavable or photochemicallycleavable moieties or linkers that can be used in place of the examplesdescribed herein. Additionally, the unique labels may also be varied,and the examples set forth herein are non-limiting. In an embodiment UVlight is used to photochemically cleave the photochemically cleavablelinkers and moieties.

This invention also provides the instant method, wherein the reversibleterminators in step (c) are 3′-O-allyl-dGTP, 3′-O-allyl-dCTP,3′-O-allyl-dATP and 3′-O-allyl-dUTP. This invention also provides theinstant method, wherein the reversible terminators in step (c) are3′-O-2-nitrobenzyl-dGTP, 3′-O-2-nitrobenzyl-dCTP,3′-O-2-nitrobenzyl-dATP and 3′-O-2-nitrobenzyl-dUTP. In an embodimentthe reversible terminator is incorporated into the growing strand ofDNA.

This invention also provides the instant method, wherein the DNApolymerase is a 9°N polymerase or a variant thereof. DNA polymeraseswhich can be used in the instant invention include, for example E. ColiDNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase™, Taq DNApolymerase and 9° N polymerase (exo-) A485L/Y409V.RNA polymerases whichcan be used in the instant invention include, for example, BacteriophageSP6, T7 and T3 RNA polymerases.

This invention also provides the instant method, wherein the DNA isbound to the solid substrate via a polyethylene glycol molecule and thesolid substrate is azide-functionalized or the DNA is immobilized on thesolid substrate via an azido linkage, an alkynyl linkage, orbiotin-streptavidin interaction; wherein (i) the four nucleotideanalogues in step (a) are 3′-O-allyl-dGTP-allyl-Cy5,3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dUTP-allyl-R6G, (ii) the four nucleotide analogues in step(a) are 3′-O-allyl-dGTP-allyl-Bodipy-FL-510,3′-O-allyl-dCTP-allyl-Bodipy-650, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dUTP-allyl-R6G, or (iii) the four nucleotide analogues instep (a) are 3′-O-allyl-dGTP-allyl-Bodipy-650,3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dUTP-allyl-R6G; and wherein the reversible terminators instep (c) are 3′-O-allyl-dGTP, 3′-O-allyl-dCTP, 3′-O-allyl-dATP and3′-O-allyl-dUTP.

This invention also provides a kit for performing the instant methodcomprising, in separate compartments,

-   -   (a) nucleotide analogues of (1) GTP, (ii) ATP, (iii) CTP        and (iv) TTP or UTP, wherein each analogue comprises (1) a base        selected from the group consisting of adenine, guanine,        cytosine, thymine or uracil, or an analogue thereof, (ii) a        deoxyribose, (iii) a cleavable moiety bound to the 3′-oxygen of        the deoxyribose and (iv) a unique label bound to the base via a        cleavable linker,    -   (b) reversible terminators comprising a nucleotide analogue        of (i) GTP, (ii) ATP, (iii) CTP and (iv) TTP or UTP, wherein        each analogue comprises (i) a base selected from the group        consisting of adenine, guanine, cytosine, thymine or uracil, or        an analogue thereof, which base does not have a unique label        bound thereto, (ii) a deoxyribose, and (iii) a cleavable moiety        bound to the 3′-oxygen of the deoxyribose;    -   (c) reagents suitable for use in DNA polymerization; and    -   (d) instructions for use.

This invention further provides the instant kit, wherein the nucleotideanalogues of part (a) are 3′-O-allyl-dGTP-allyl-Cy5,3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dUTP-allyl-R6G. This invention further provides the instantkit, wherein the nucleotide analogues of part (a) are3′-O-allyl-dGTP-allyl-Bodipy-FL-510, 3′-O-allyl-dCTP-allyl-Bodipy-650,3′-O-allyl-dATP-allyl-ROX and 3′-O-allyl-dUTP-allyl-R6G. This inventionfurther provides the instant kit, wherein the nucleotide analogues ofpart (a) are 3′-O-allyl-dGTP-allyl-Bodipy-650,3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dUTP-allyl-R6G. This invention further provides the instantkit, wherein the nucleotide analogues of part (b) are 3′-O-allyl-dGTP,3′-O-allyl-dCTP, 3′-O-allyl-dATP and 3′-O-allyl-dUTP. This inventionfurther provides the instant kit, the reversible terminators in step (c)are 3′-O-2-nitrobenzyl-dGTP, 3′-O-2-nitrobenzyl-dCTP,3′-O-2-nitrobenzyl-dATP and 3′-O-2-nitrobenzyl-dUTP.

The methods and kits of this invention may be applied, mutatis mutandis,to the sequencing of RNA, or to determining the identity of aribonucleotide.

Methods for production of cleavably capped and/or cleavably linkednucleotide analogues are disclosed in U.S. Pat. No. 6,664,079, which ishereby incorporated by reference. Combinatorial fluorescence energy tagsand methods for production thereof are disclosed in U.S. Pat. No.6,627,748, which is hereby incorporated by reference.

In an embodiment, the DNA or nucleic acid is attached/bound to the solidsurface by covalent site-specific coupling chemistry compatible withDNA.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

DNA sequencing by synthesis (SBS) on a solid surface during polymerasereaction offers a new paradigm to decipher DNA sequences. Disclosed hereis the construction of such a novel DNA sequencing system usingmolecular engineering approaches. In this approach, four nucleotides (A,C, G, T) are modified as reversible terminators by attaching a cleavablefluorophore to the base and capping the 3′-OH group with a smallchemically reversible moiety so that they are still recognized by DNApolymerase as substrates. It is found that an allyl moiety can be usedsuccessfully as a linker to tether a fluorophore to 3′-O-allyl-modifiednucleotides, forming chemically cleavable fluorescent nucleotidereversible terminators, 3′-O-allyl-dNTPs-allyl-fluorophore, forapplication in SBS. The fluorophore and the 3′-O-allyl group on a DNAextension product, which is generated by incorporating3′-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removedsimultaneously in 30 seconds by Pd-catalyzed deallylation in aqueousbuffer solution. This one-step dual-deallylation reaction thus allowsthe re-initiation of the polymerase reaction and increases the SBSefficiency. DNA templates consisting of homopolymer regions wereaccurately sequenced by using this new class of fluorescent nucleotideanalogues on a DNA chip and a 4-color fluorescent scanner.

It is known that some modified DNA polymerases are highly tolerable fornucleotides with extensive modifications with bulky groups such asenergy transfer dyes at the 5-position of the pyrimidines (T and C) and7-position of purines (G and A) (23, 24). The ternary complexes of a ratDNA polymerase, a DNA template-primer, and dideoxycytidine triphosphatehave been determined (22) which supports this fact. It was hypothesizrfthat if a unique fluorescent dye is linked to the 5-position of thepyrimidines (T and C) and the 7-position of purines (G and A) via acleavable linker, and a small chemical moiety is used to cap the 3′-OHgroup, then the resulting nucleotide analogues may be able toincorporate into the growing DNA strand as terminators. Based on thisrationale, SBS approach was conceived using cleavable fluorescentnucleotide analogues as reversible terminators to sequencesurface-immobilized DNA in 2000 (FIG. 1) (25). In this approach, thenucleotides are modified at two specific locations so that they arestill recognized by DNA polymerase as substrates: (i) a differentfluorophore with a distinct fluorescent emission is linked to each ofthe 4 bases through a cleavable linker and (ii) the 3′-OH group iscapped by a small chemically reversible moiety. DNA polymeraseincorporates only a single nucleotide analogue complementary to the baseon a DNA template covalently linked to a surface. After incorporation,the unique fluorescence emission is detected to identify theincorporated nucleotide and the fluorophore is subsequently removed. The3′-OH group is then chemically regenerated, which allows the next cycleof the polymerase reaction to proceed. Since the large surface on a DNAchip can have a high density of different DNA templates spotted, eachcycle can identify many bases in parallel, allowing the simultaneoussequencing of a large number of DNA molecules. The feasibility ofperforming SBS on a chip using 4 photocleavable fluorescent nucleotideanalogues was previously established (26) and it was discovered that anallyl group can be used as a cleavable linker to bridge a fluorophore toa nucleotide (27). The design and synthesis of two photocleavablefluorescent nucleotides as reversible terminators for polymerasereaction has already been reported (28, 29).

Previous research efforts in the present laboratory have firmlyestablished the molecular level strategy to rationally modify thenucleotides by attaching a cleavable fluorescent dye to the base andcapping the 3′-OH with a small chemically reversible moiety for SBS.This approach was recently adopted by Genomics Industry to potentiallyprovide a new platform for DNA sequencing (30). Here the design andsynthesis of 4 chemically cleavable fluorescent nucleotide analogues asreversible terminators for SBS is disclosed. Each of the nucleotideanalogues contains a 3′-O-allyl group and a unique fluorophore with adistinct fluorescence emission at the base through a cleavable allyllinker.

It was first established that these nucleotide analogues are goodsubstrates for DNA polymerase in a solution-phase DNA extension reactionand that the fluorophore and the 3′-O-allyl group can be removed withhigh efficiency in aqueous solution. Then SBS was performed using these4 chemically cleavable fluorescent nucleotide analogues as reversibleterminators to identify ˜20 continuous bases of a DNA templateimmobilized on a chip. Accurate DNA sequences were obtained for DNAtemplates containing homopolymer sequences. The DNA template wasimmobilized on the surface of the chip that contains a PEG linker with1,3-dipolar azide-alkyne cycloaddition chemistry. These resultsindicated that successful cleavable fluorescent nucleotide reversibleterminators for 4-color DNA sequencing by synthesis can be designed byattaching a cleavable fluorophore to the base and capping the 3′-OH witha small chemically reversible moiety so that they are still recognizedby DNA polymerase as substrates. Further optimization of the approachwill lead to even longer sequencing readlengths.

Design and Synthesis of Chemically Cleavable Fluorescent NucleotideAnalogues as Reversible Terminators for SBS

To demonstrate the feasibility of carrying out de novo DNA sequencing bysynthesis on a chip, four chemically cleavable fluorescent nucleotideanalogues (3′-O-allyl-dCTP-allyl-Bodipy-FL-510,3′-O-allyl-dUTP-allyl-R6G, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dGTP-allyl-Bodipy-650/Cy5) (FIG. 2) were designed andsynthesized as reversible terminators for DNA polymerase reaction.Modified DNA polymerases have been shown to be highly tolerant tonucleotide modifications with bulky groups at the 5-position ofpyrimidines (C and U) and the 7-position of purines (A and G). Thus,each unique fluorophore was attached to the 5 position of C/U and the 7position of A/G through an allyl carbamate linker. However, due to theclose proximity of the 3′ position on the sugar ring of a nucleotide tothe amino acid residues of the active site of the DNA polymerase, arelatively small allyl moiety was chosen as the 3′-OH reversible cappinggroup. It was found that the fluorophore and the 3′-O-allyl group on aDNA extension product, which is generated by incorporation of thechemically cleavable fluorescent nucleotide analogues, are removedsimultaneously in 30 seconds by Pd-catalyzed deallylation in aqueoussolution. This one-step dual-deallylation reaction thus allows there-initiation of the polymerase reaction. The detailed synthesisprocedure and characterization of the 4 novel nucleotide analogues inFIG. 2 are described in

Materials and Methods

In order to verify that these fluorescent nucleotide analogues areincorporated accurately in a base-specific manner in a polymerasereaction, four continuous steps of DNA extension and deallylation werecarried out in solution. This allows the isolation of the DNA product ateach step for detailed molecular structure characterization by MALDI-TOFmass spectrometry (MS) as shown in FIG. 3. The first extension product5′-U(allyl-R6G)-3′-O-allyl (1) was purified by HPLC and analyzed usingMALDI-TOF MS [FIG. 3(A)]. This product was then deallylated using aPd-catalyzed deallylation cocktail [1× Thermopol I reactionbuffer/Na₂PdCl₄/P(PhSO₃Na)₃]. The active Pd catalyst is generated fromNa₂PdCl₄ and a ligand P(PhSO₃Na)₃ (TPPTS) to mediate the deallylationreaction in DNA compatible aqueous condition to simultaneously cleaveboth the fluorophore and the 3′-O-allyl group (28). The deallylatedproduct (2) was also analyzed using MALDI-TOF MS [FIG. 3(B)]. As can beseen from FIG. 3(A), the MALDI-TOF MS spectrum consists of a distinctpeak at m/z 6469 corresponding to the DNA extension product5′-U(allyl-R6G)-3′-O-allyl (1), which confirms that the nucleotideanalogue can be incorporated base specifically among pool of all four(A, C, G, T) by DNA polymerase into a growing DNA strand. FIG. 3(B)shows the deallylation result of the above DNA product. The peak at m/z6469 has completely disappeared while the peak corresponding to the dualdeallylated product 5′-U (2) appears as the sole dominant peak at m/z5870, which establishes that the Pd-catalyzed deallylation reactioncompletely cleaves both the fluorophore and the 3′-O-allyl group withhigh efficiency without damaging the DNA. The next extension reactionwas carried out using this deallylated DNA product with a free 3′-OHgroup regenerated as a primer along with four allyl modified fluorescentnucleotide mixture to yield an extension product5′-UG(allyl-Bodipy-650)-3′-O-allyl (3). As described above, theextension product 3 was analyzed by MALDI-TOF MS producing a dominantpeak at m/z 6984 [FIG. 3(C)], and then deallylated for further MSanalysis yielding a single peak at m/z 6256 (product 4) [FIG. 3(D)]. Thethird extension reaction yielding 5′-UGA(allyl-ROX)-3′-O-allyl (5), thefourth extension reaction yielding5′-UGAC(allyl-Bodipy-FL-510)-3′-O-allyl (7) and their deallylationreactions to yield products 6 and 8 were similarly carried out andanalyzed by MALDI-TOF MS as shown in FIGS. 3(E), 3(F), 3(G) and 3(H).The chemical structures of the extension and cleavage products for eachstep are shown in FIG. 4. These results demonstrate that theabove-synthesized 4 chemically cleavable fluorescent nucleotideanalogues are successfully incorporated with high fidelity into thegrowing DNA strand in a polymerase reaction, and furthermore, both thefluorophore and the 3′-O-allyl group are efficiently removed by using aPd-catalyzed deallylation reaction, which makes it feasible to use themfor SBS on a chip.

4-Color DNA Sequencing with Chemically Cleavable Fluorescent NucleotideAnalogues as Reversible Terminators on a DNA Chip

The chemically cleavable fluorescent nucleotide analogues were then usedin an SBS reaction to identify the sequence of the DNA templateimmobilized on a solid surface. A site-specific 1,3-dipolarcycloaddition coupling chemistry was used to covalently immobilize thealkyne-labeled self-priming DNA template on the azido-functionalizedsurface in the presence of a Cu(I) catalyst. The principal advantageoffered by the use of a self-priming moiety as compared to usingseparate primers and templates is that the covalent linkage of theprimer to the template in the self-priming moiety prevents any possibledissociation of the primer from the template during the process of SBS.To prevent non-specific absorption of the unincorporated fluorescentnucleotides on the surface of the chip, a PEG linker is introducedbetween the DNA templates and the chip surface (FIG. 5). This approachwas shown to produce very low background fluorescence after cleavage toremove the fluorophore as demonstrated by the DNA sequencing datadescribed below.

SBS was first performed on a chip-immobilized DNA template that has nohomopolymer sequences using the four chemically cleavable fluorescentnucleotide reversible terminators (3′-O-allyl-dCTP-allyl-Bodipy-FL-510,3′-O-allyl-dUTP-allyl-R6G, 3′-O-allyl-dATP-allyl-ROX and3′-O-allyl-dGTP-allyl-Cy5) and the results are shown in FIG. 6. Thestructure of the self-priming DNA moiety is shown schematically in FIG.6A, with the first 13 nucleotide sequences immediately after the primingsite. The de novo sequencing reaction on the chip was initiated byextending the self-priming DNA using a solution containing all four3′-O-allyl-dNTPs-allyl-fluorophore, and a 9° N mutant DNA polymerase. Inorder to negate any lagging fluorescence signal that is caused bypreviously unextended priming strand, a synchronization step was addedto reduce the amount of unextended priming strands after the extensionwith the fluorescent nucleotides. A synchronization reaction mixtureconsisting of all four 3′-O-allyl-dNTPs (FIG. 7), which have a higherpolymerase incorporation efficiency due to the lack of a fluorophorecompared to the bulkier 3′-O-allyl-dNTPs-allyl-fluorophore, was usedalong with the 9° N mutant DNA polymerase to extend any remainingpriming strand that has a free 3′-OH group to synchronize theincorporation. The extension by 3′-O-allyl-dNTPs also enhances theenzymatic incorporation of the next nucleotide analogue, because aftercleavage to remove the 3′-O-allyl group, the DNA product extended by3′-O-allyl-dNTPs carry no modification groups. After washing, theextension of the primer by only the complementary fluorescent nucleotidewas confirmed by observing a red signal (the emission from Cy5) in a4-color fluorescent scanner [FIG. 6B (1)]. After detection of thefluorescent signal, the chip surface was immersed in a deallylationcocktail [1× Thermolpol I reaction buffer/Na₂PdCl₄/P(PhSO₃Na)₃] andincubated for 5 min at 60° C. to cleave both the fluorophore and3′-O-allyl group simultaneously. The chip was then immediately immersedin a 3 M Tris-HCl buffer (pH 8.5) and incubated for 5 min at 60° C. toremove the Pd complex. The surface was then washed, and a negligibleresidual fluorescent signal was detected to confirm cleavage of thefluorophore. This was followed by another extension reaction using3′-O-allyl-dNTPs-allyl-fluorophore mixture to incorporate the nextfluorescent nucleotide complementary to the subsequent base on thetemplate. The entire process of incorporation, synchronization,detection and cleavage was performed multiple times using the fourchemically cleavable fluorescent nucleotide reversible terminators toidentify 13 successive bases in the DNA template. The fluorescence imageof the chip for each nucleotide addition is shown in FIG. 6B, while aplot of the fluorescence intensity vs. the progress of sequencingextension (raw 4-color sequencing data) is shown in FIG. 6C. The DNAsequences are unambiguously identified from the 4-color raw fluorescencedata without any processing.

Comparison of 4-Color SBS with Pyrosequencing.

To further verify the advantage of SBS method using the four chemicallycleavable fluorescent nucleotide reversible terminators, we carried outsimilar sequencing reaction as described above on a DNA template whichcontained two separate homopolymeric regions (stretch of 10 T's and 5T's) as shown in FIG. 8 (panel A). These sequencing raw data wereproduced by adding all 4 fluorescent nucleotide reversible terminatorstogether to the DNA template immobilized on the chip followed bysynchronization reaction with four 3′-O-allyl-dNTPs, detecting theunique fluorescence emission for sequence determination, then cleavingthe fluorophore and the 3′-O-allyl group in one step to continue thesequencing cycles. All 20 bases including the individual base (A, T, C,G), the 10 repeated A's and the 5 repeated A's are clearly identified.The small groups of peaks between the identified bases are fluorescentbackground from the DNA chip, which does not build up as the cyclecontinues. Panel B in FIG. 8 shows the pyrosequencing data of the sameDNA template containing the homopolymeric sequences. The first 4individual bases are clearly identified. The two homopolymeric regions(10 A's) and (5 A's) produce two large peaks, but it is very difficultto identify the exact sequence from the data.

CONCLUSION

Four novel chemically cleavable fluorescent nucleotide analogues havebeen synthesized and characterized and have been used to produce a4-color de novo DNA sequencing data on a chip. In doing so, two criticalrequirements for using SBS method to sequence DNA have been achievedunambiguously. First, a strategy to use a chemically reversible moietyto cap the 3′-OH group of the nucleotide has been successfullyimplemented so that the nucleotide terminates the polymerase reaction toallow the identification of the incorporated nucleotide. In additionthese reversible terminators allow for the addition of all fournucleotides simultaneously in performing SBS. This ultimately reducesthe number of cycles needed to complete the sequencing cycle, increasessequencing accuracy due to competition of the 4 nucleotides in thepolymerase reaction, and enables accurate determination of homopolymericregions of DNA template. Second, efficient removal of both thefluorophore and the 3′-OH capping group after the fluorescence signaldetection have successfully been carried out which increases the overallefficiency of SBS.

The key factor governing the sequencing readlength of our 4-color SBSapproach is the stepwise yield that are determined by the nucleotideincorporation efficiency and the yield of the cleavage of thefluorophore and the 3′-OH capping group from the DNA extension products.This stepwise yield here is ˜ 99% based on measurement of the DNAproducts in solution phase. The yield on the surface is difficult tomeasure precisely due to fluctuations in the fluorescence imaging usingthe current manual fluorescent scanner. The strong fluorescence signaleven for the 20^(th) base shown in FIG. 8 indicates that we should beable to extend the readlength even further. In terms of readlength,Sanger sequencing is still the gold standard with readlength of over 800bp but limited in throughput and cost. The readlength of pyrosequencingis ˜100 bp but with high error rate due to difficulty in accuratelydetermining the sequences of homopolymers. The 4-color SBS readlength ona manual fluorescent scanner is currently at ˜20 bp with high accuracy.This readlength will increase with automation of the extension, cleavageand washing steps. The DNA polymerases and fluorescent labeling used inthe automated 4-color Sanger sequencing method have undergone almost twodecades of consistent incremental improvements after the basicfluorescent Sanger methods were established (2, 3). Following the sameroute, it is expected that the basic principle and strategy outlined inour 4-color SBS method will stimulate further improvement of thesequencing by synthesis methodology with engineering of high performancepolymerases tailored for the cleavable fluorescent nucleotideterminators and testing alternative linkers and 3′-OH reversible cappingmoiety. It has been well established that using emulsion PCR onmicrobeads, millions of different DNA templates are immobilized on asurface of a chip (11, 16). This high density DNA templates coupled withour 4-color SBS approach will generate a high-throughput (>20 millionsbases/chip) and high-accurate platform for a variety of sequencing anddigital gene expression analysis projects.

Materials and Methods

¹H NMR spectra were recorded on a Bruker DPX-400 (400 MHz) spectrometerand reported in ppm from a CD₃OD or DMSO-d₆ internal standard (3.31 or2.50 ppm respectively). Data were reported as follows: (s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublets,ddd=doublet of doublets of doublets; coupling constant(s) in Hz;integration; assignment). Proton decoupled ¹³C NMR spectra were recordedon a Bruker DPX-400 (100 MHz)

Synthetic scheme to prepare the chemically cleavable fluorescentnucleotides. a, DMF/1 M NaHCO₃ solution; b, N, N′-disuccinimidylcarbonate (DSC), triethylamine; c, 0.1 M NaHCO₃/Na₂CO₃ aqueous buffer(pH 8.7) spectrometer and reported in ppm from a CD₃OD, DMSO-de or CDCl₃internal standard (49.0, 39.5 or 77.0 ppm respectively). Protondecoupled ³¹P NMR spectra were recorded on a Bruker DPX-300 (121.4 MHz)spectrometer and reported in ppm from an 85% H₃PO₄ external standard.High Resolution Mass Spectra (HRMS) were obtained on a JEOL JMS HX 110Amass spectrometer. Compounds 30 and 32 were purchased from Berry &Associates. All Dye NHS esters were purchased from Molecular Probes andGE Healthcare. All other chemicals were purchased from Sigma-Aldrich.

I. Synthesis of 3′-O-allyl-dNTPs-allyl-Fluorophore

Chemically cleavable fluorescent nucleotides3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dUTP-allyl-R6G,3′-O-allyl-dATP-allyl-ROX, 3′-O-allyl-dGTP-allyl-Bodipy-650 and3′-O-allyl-dGTP-allyl-Cy5 were synthesized according to the aboveScheme. A chemically cleavable linker 4-amino-2-methylene-1-butanol(allyl-linker) was reacted with the N-hydroxysuccinimide (NHS) ester ofthe corresponding fluorescent dye to produce an intermediateallyl-fluorophore, which was converted to an allyl-fluorophore NHS esterby reacting with N,N′-disuccinimidyl carbonate. The coupling reactionbetween the different allyl-fluorophore NHS esters and3′-O-allyl-dNTPs-NH₂ produced the four chemically cleavable fluorescentnucleotides.

1. Synthesis of Allyl-Linker

2-Triphenylmethoxymethyl-2-propen-1-ol (2). To a stirred solution oftrityl chloride (4.05 g; 14.3 mmol) and 2-methylenepropane-1,3-diol 1(1.20 mL; 14.3 mmol) in dry CH₂Cl₂ (20 mL), triethylamine (4.0 mL; 28.5mmol) was added slowly at room temperature. The mixture was stirred atroom temperature for 1 h, and then ethyl acetate (100 mL) and saturatedaqueous NaHCO₃ (30 mL) were added. The organic phase was washed withsaturated aqueous NaHCO₃, NaCl, and dried over anhydrous Na₂SO₄. Afterevaporation, the residue was purified by flash column chromatographyusing ethyl acetate-hexane (1:10˜5) as the eluent to afford 2 as a whitesolid (2.89 g; 62% yield): ¹H NMR (400 MHz, CDCl₃) δ 7.42-7.48 (m, 6H,six of ArH), 7.27-7.33 (m, 6H, six of ArH), 7.20-7.27 (m, 3H, three ofArH), 5.26 (s, 1H, one of C═CH₂), 5.17 (s, 1H, one of C═CH₂), 4.13 (d,J=6.1 Hz, 2H, CH₂OH), 3.70 (s, 2H, Ph₃COCH₂), 1.72 (t, J=6.1 Hz, 1H,CH₂OH); ¹³C NMR (100 MHz, CDCl₃) δ 145.4, 143.6, 128.3, 127.6, 126.8,111.6, 87.0, 65.3, 64.5.

1-Bromo-2-triphenylmethoxymethyl-2-propene (3). To a stirred solution of2 (2.56 g; 7.74 mmol) in CH₂Cl₂ (75 ml), CBr₄ (3.63 g; 10.83 mmol) andtriphenylphosphine (2.47 g; 9.31 mmol) were added respectively at 0° C.The mixture was stirred at room temperature for 40 min. Ethyl acetate(100 mL) and saturated aqueous NaHCO₃ (30 mL) were added at 0° C. Theorganic phase was washed with saturated aqueous NaCl, and dried overanhydrous Na₂SO₄. After evaporation, the residue was purified by flashcolumn chromatography using CH₂Cl₂-hexane (1:5) as the eluent to afford3 as white solid (3.02 g; 92% yield): ¹H NMR (400 MHz, CDCl₃) δ7.42-7.48 (m, 6H, six of ArH), 7.27-7.33 (m, 6H, six of ArH), 7.20-7.27(m, 3H, three of ArH), 5.37 (s, 1H, one of C═CH₂), 5.31 (s, 1H, one ofC═CH₂), 4.01 (s, 2H, CH₂Br), 3.75 (s, 2H, Ph₃COCH₂); ¹³C NMR (100 MHz,CDCl₃) δ 143.6, 142.6, 128.4, 127.6, 126.9, 115.8, 86.9, 64.2, 33.5.

3-Triphenylmethoxymethyl-3-butenonitrile (4). To a stirred solution of 3(1.45 g; 3.69 mmol) and in dry CH₃CN (37 mL), trimethylsilyl cyanide(0.49 mL; 3.69 mmol) was added. Then, 1 M tetrabutylammonium fluoride(TBAF) in THF solution (3.69 mL, 3.69 mmol) was added into the abovereaction mixture slowly at room temperature. The mixture was stirred for20 min. After evaporation, the residue was diluted with ethyl acetate(100 mL) and saturated aqueous NaHCO₃ (30 mL). The organic phase waswashed with saturated aqueous NaCl and dried over anhydrous Na₂SO₄.After evaporation, the residue was purified by flash columnchromatography using acetate-hexane (1:10) as the eluent to afford 4 aswhite solid (1.01 g; 64% yield): ¹H NMR (400 MHz, CDCl₃) δ 7.39-7.45 (m,6H, six of ArH), 7.21-7.34 (m, 9H, nine of ArH), 5.31 (s, 2H, C═CH₂),3.64 (s, 2H, Ph₃COCH₂), 3.11 (s, 2H, CH₂CN); ¹³C NMR (100 MHz, CDCl₃) δ143.3, 135.5, 128.2, 127.7, 126.9, 116.8, 114.7, 87.0, 65.7, 21.9.

3-Triphenylmethoxymethyl-3-buten-1-amine (5). To a stirred solution ofLiAlH₄ (119 mg; 2.98 mmol) in dry ether (5 mL), AlCl₃ (400 mg; 2.94mmol) was added slowly at 0° C. and the mixture was stirred for 15 min.The mixture of 4 (829 mg; 2.44 mmol) in dry ether (9 mL) was added andthen continued to stir at 0° C. for another 3 h. Afterwards, 10% aqueousNaOH (10 mL) was added to quench the reaction. The organic phase waswashed with saturated aqueous NaHCO₃, NaCl, and dried over anhydrousK₂CO₃. After evaporation, the residue was further purified by flashcolumn chromatography using CH₃OH—CH₂Cl₂ (1:20˜5) as the eluent toafford 5 as colorless oil (545 mg; 65% yield): ¹H NMR (400 MHz, CDCl₃) δ7.41-7.48 (m, 6H, six of ArH), 7.26-7.33 (m, 6H, six of ArH), 7.19-7.26(m, 3H, three of ArH), 5.33 (s, 1H, one of C═CH₂), 4.96 (s, 1H, one ofC═CH₂), 3.53 (s, 2H, Ph₃COCH₂), 2.70 (m, 2H, CH₂CH₂NH₂), 2.18 (t, J=6.7Hz, 2H, CH₂CH₂NH₂), 2.06 (br s, 2H, NH₂); ¹³C NMR (100 MHz, CDCl₃) δ143.6, 143.4, 128.1, 127.4, 126.5, 111.3, 86.5, 66.1, 39.8, 37.4; HRMS(FAB+) calcd for C₂₄H₂₆ON (M+H⁺): 344.2014, found: 344.2017.

4-Amino-2-methylene-1-butanol (6). To a stirred solution of 5 (540 mg;1.57 mmol) in CH₃OH (11 mL), HCl (2M solution in ether; 5.5 mL) wasadded at room temperature and the mixture was stirred for 1 h. Then 7Mammonia in CH₃OH solution (2.7 mL) was added into the above mixture atroom temperature and continued to stir for another 10 min. Afterfiltration, the solid was washed with CH₃OH and combined with thefiltrate. After evaporation, the crude product was further purified byflash column chromatography using CH₃OH—CH₂Cl₂ (1:4) as the eluent toafford 6 as colorless oil (151 mg; 95% yield): ¹H NMR (400 MHz, CD₃OD)δ5.19 (s, 1H, one of C═CH₂), 5.01 (s, 1H, one of C═CH₂), 4.06 (s, 2H,CH₂OH), 3.10 (t, J=7.5 Hz, 2H, CH₂CH₂NH₂), 2.46 (t, J=7.5 Hz, 2H,CH₂CH₂NH₂); ¹³C NMR (100 MHz, CD₃OD) δ 145.3, 113.7, 65.5, 39.5, 32.0;MS (FAB+) calcd for C₅H₁₂ON (M+H⁺): 102.09, found: 102.12.

2. Synthesis of Allyl-Fluorophore

A general procedure for the synthesis of Allyl-Fluorophore is asfollows. To a stirred solution of 6 (3.5 mg; 34.6 μmol) in DMF (450 μL),aqueous NaHCO₃ (1M solution; 100 μL) was added at room temperature. Themixture was stirred for 5 min. Dye NHS (N-hydroxysuccinimide) ester (5mg) in DMF (450 μL) was added and then the mixture was stirred at roomtemperature for 6 h. The crude product was further purified by apreparative TLC plate using CH₃OH—CH₂Cl₂ as the eluent to affordAllyl-Fluorophore.

Allyl-Bodipy-FL-510 (7). ¹H NMR (400 MHz, CD₃OD) δ 7.42 (s, 1H), 7.00(d, J=4.0 Hz, 1H), 6.32 (d, J=4.0 Hz, 1H), 6.21 (s, 1H), 5.06 (s, 1H,one of C═CH₂), 4.87 (s, 1H, one of C═CH₂, partly superimposed by solventsignal), 4.01 (s, 2H, CH₂OH), 3.33 (t, J=7.5 Hz, 2H, partly superimposedby solvent signal), 3.21 (t, J=7.7 Hz, 2H), 2.59 (t, J=7.7 Hz, 2H), 2.51(s, 3H, one of ArCH₃), 2.28 (s, 3H), 2.26 (t, J=7.1 Hz, 2H); HRMS (FAB+)calcd for C₁₉H₂₄O₂N₃F₂B (M): 375.1933, found: 375.1957.

Allyl-R6G (8). ¹H NMR (400 MHz, CD₃OD) δ 8.12 (d, J=8.1 Hz, 1H), 8.05(dd, J=1.8, 8.1 Hz, 1H), 7.66 (d, J=1.6 Hz, 1H), 7.02 (s, 2H), 6.88 (s,2H), 5.08 (s, 1H, one of C═CH₂), 4.92 (s, 1H, one of C═CH₂), 4.06 (s,2H, CH₂OH), 3.48-3.56 (m, 6H), 2.40 (t, J=7.2 Hz, 2H), 2.13 (s, 6H),1.36 (t, J=7.2 Hz, 6H); HRMS (FAB+) calcd for C₃₂H₃₆O₅N₃ (M+H⁺):542.2655, found: 542.2648.

Allyl-ROX (9). ¹H NMR (400 MHz, CD₃OD) δ 8.03 (d, J=8.1 Hz, 1H), 7.98(dd, J=1.6, 8.1 Hz, 1H), 7.60 (d, J=1.4 Hz, 1H), 6.75 (s, 2H), 5.08 (s,1H, one of C═CH₂), 4.91 (s, 1H, one of C═CH₂), 4.05 (s, 2H, CH₂OH),3.45-3.57 (m, 10H), 3.03-3.10 (m, 4H), 2.64-2.73 (m, 4H), 2.38 (t, J=7.1Hz, 2H), 2.04-2.15 (m, 4H), 1.89-1.99 (m, 4H); HRMS (FAB+) calcd forC₃₈H₄₀O₅N₃ (M+H⁺): 618.2968, found: 618.2961.

Allyl-Bodipy-650 (10). ¹H NMR (400 MHz, CD₃OD) δ 8.12 (dd, J=0.9, 3.8Hz, 1H), 7.63 (m, 3H), 7.54 (d, J=6.4 Hz, 2H), 7.35 (s, 1H), 7.18-7.22(m, 2H), 7.12 (m, 2H), 7.06 (d, J=8.8 Hz, 2H), 6.85 (d, J=4.2 Hz, 1H),5.06 (s, 1H, one of C═CH₂), 4.86 (s, 1H, one of C═CH₂), 4.56 (s, 2H),4.00 (s, 2H, CH₂OH), 3.28 (m, 4H), 2.23 (t, J=7.1 Hz, 2H), 2.14 (t,J=7.5 Hz, 2H), 1.49-1.62 (m, 4H), 1.25-1.34 (m, 2H); HRMS (FAB+) calcdfor C₃₄H₃₇O₄N₄F₂SB (M⁺): 646.2603, found: 646.2620.

Allyl-Cy5 (11). ¹H NMR (400 MHz, CD₃OD) δ 7.75-7.86 (m, 2H), 7.43-7.62(m, 3H), 6.65 (m, 1H), 6.25-6.53 (m, 5H), 5.06 (s, 1H, one of C═CH₂),4.86 (s, 1H, one of C═CH₂), 4.56 (s, 2H), 4.00 (s, 2H, CH₂OH), 3.28 (m,6H), 2.03-2.40 (m, 4H), 1.55 (t, J=7.2 Hz, 3H), 1.31 (s, 6H), 1.26 (s,6H), 1.25-1.64 (m, 6H); HRMS (FAB+) calcd for C₃₈H₄₈O₈N₃S₂ (M⁺):738.2888, found: 738.2867.

3. Synthesis of Allyl-Fluorophore NHS Ester

A general procedure for the synthesis of Allyl-Dye NHS ester is asfollows. To a stirred solution of Allyl-Fluorophore (4 mg) in dry DMF(350 μL), DSC (8.0 mg; 31.2 μmol) and triethylamine (5.0 μL; 35.4 μmol)were added respectively. The reaction mixture was stirred at roomtemperature for 10 h. After evaporation, the crude product was furtherpurified by flash column chromatography using CH₃OH—CH₂Cl₂ as the eluentto afford Allyl-Fluorophore NHS ester, which was used directly for thenext step.

4. Synthesis of 3′-O-allyl-dCTP-allyl-Bodipy-FL-510 (23)

5′-O-(tert-Butyldimethylsilyl)-5-iodo-2′-deoxycytidine (18). To astirred mixture of 17 (1.00 g; 2.83 mmol) and imidazole (462 mg; 6.79mmol) in anhydrous DMF (14.0 mL), tert-butyldimethylsilyl chloride(TBDMSCl) (510 mg; 3.28 mmol) was added. The reaction mixture wasstirred at room temperature for 20 h. After evaporation, the residue waspurified by flash column chromatography using CH₃OH—CH₂Cl₂ (1:20) as theeluent to afford 18 as white solid (1.18 g; 89% yield): ¹H NMR (400 MHz,CD₃OD) δ 8.18 (s, 1H, 6-H), 6.17 (dd, J=5.8, 7.5 Hz, 1H, 1′-H), 4.34 (m,1H, 3′-H), 4.04 (m, 1H, 4′-H), 3.93 (dd, J=2.5, 11.6 Hz, 1H, one of5′-H), 3.84 (dd, J=2.9, 11.6 Hz, 1H, one of 5′-H), 2.41-2.48 (ddd,J=2.5, 5.8, 13.5 Hz, 1H, one of 2′-H), 2.01-2.08 (ddd, J=5.9, 7.6, 13.5Hz, 1H, one of 2′-H), 0.95 (s, 9H, C(CH₃)₃), 0.17 (s, 3H, one of SiCH₃),0.16 (s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 165.5, 156.8,147.8, 89.4, 88.3, 72.8, 64.6, 57.1, 43.1, 26.7, 19.4, −4.8, −4.9; HRMS(FAB+) calcd for C₁₅H₂₇O₄N₃SiI (M+H⁺): 468.0816, found: 468.0835.

3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-5-iodo-2′-deoxycytidine (19).To a stirred solution of 18 (1.18 g; 2.52 mmol) in anhydrous THF (43mL), 95% NaH powder (128 mg; 5.07 mmol) was added. The suspensionmixture was stirred at room temperature for 45 min. Allyl bromide (240μL, 2.79 mmol) was then added at 0° C. and the reaction was stirred atroom temperature for another 14 h with exclusion of moisture. Saturatedaqueous NaHCO₃ (10 mL) was added at 0° C. and the reaction mixture wasstirred for 10 min. After evaporation, the residue was dissolved inethyl acetate (150 mL). The solution was then washed with saturatedaqueous NaHCO₃ and NaCl, and dried over anhydrous Na₂SO₄. Afterevaporation, the residue was purified by flash column chromatographyusing ethyl acetate as the eluent to afford 19 as white solid (601 mg;47% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.15 (s, 1H, 6-H), 6.12 (dd,J=5.6, 8.0 Hz, 1H, 1′-H), 4.17 (m, 1H, 4′-H), 4.14 (m, 1H, 3′-H),3.98-4.10 (m, 2H, CH₂CH═CH₂), 3.93 (dd, J=2.8, 11.5 Hz, 1H, one of5′-H), 3.83 (dd, J=2.8, 11.5 Hz, 1H, one of 5′-H), 2.53-2.60 (ddd,J=1.7, 5.6, 13.6 Hz, 1H, one of 2′-H), 1.94-2.02 (ddd, J=5.9, 8.0, 13.6Hz, 1H, one of 2′-H), 0.94 (s, 9H, C(CH₃)₃), 0.17 (s, 3H, one of SiCH₃),0.16 (s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 165.4, 156.7,147.7, 135.5, 117.2, 88.2, 87.0, 80.4, 70.9, 64.8, 57.3, 40.1, 26.7,19.4, −4.7, −4.9; HRMS (FAB+) calcd for C₁₈H₃₁O₄N₃SiI (M+H⁺): 508.1129,found: 508.1123.

3′-O-Allyl-5-iodo-2′-deoxycytidine (20). To a stirred solution of 19(601 mg; 1.18 mmol) in anhydrous THF (28 mL), 1 M TBAF in THF solution(1.31 mL; 1.31 mmol) was added and the reaction mixture was stirred atroom temperature for 1 h. After evaporation, the residue was dissolvedin ethyl acetate (100 mL). The solution was then washed with saturatedaqueous NaCl and dried over anhydrous Na₂SO₄. After evaporation, theresidue was purified by flash column chromatography using CH₃OH—CH₂Cl₂(1:10) as the eluent to afford 20 as white crystals (329 mg; 71% yield):¹H NMR (400 MHz, CD₃OD) δ 8.47 (s, 1H, 6-H), 6.15 (dd, J=6.2, 6.7 Hz,1H, 1′-H), 5.87-5.98 (m, 1H, CH₂CH═CH₂), 5.26-5.33 (dm, J=17.2 Hz, 1H,one of CH₂CH═CH₂), 5.14-5.19 (dm, J=10.5 Hz, 1H, one of CH₂CH═CH₂), 4.18(m, 1H, 3′-H), 4.08 (m, 1H, 4′-H), 3.98-4.10 (m, 2H, CH₂CH═CH₂), 3.82(dd, J=3.2, 13.0 Hz, 1H, one of 5′-H), 3.72 (dd, J=3.3, 13.0 Hz, 1H, oneof 5′-H), 2.44-2.51 (ddd, J=3.2, 6.0, 13.6 Hz, 1H, one of 2′-H),2.07-2.15 (m, 1H, one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ 165.4, 156.9,148.8, 135.6, 117.0, 87.9, 86.9, 79.6, 71.2, 62.7, 57.2, 39.7; HRMS(FAB+) calcd for C₁₂H₁₇O₄N₃I (M+H⁺): 394.0264, found: 394.0274.

3′-O-Allyl-5-{3-[(trifluoroacetyl)amino]prop-1-ynyl}-2′-deoxycytidine(21). To a stirred solution of 20 (329 mg; 0.84 mmol) in anhydrous DMF(3.7 mL), tetrakis(triphenylphosphine)palladium(0) (97 mg; 0.084 mmol)and CuI (35 mg; 0.18 mmol) were added. The reaction mixture was stirredat room temperature for 10 min. Then N-propargyltrifluoroacetamide (379mg; 2.51 mmol) and Et₃N (233 μL; 1.68 mmol) were added into the abovereaction mixture. The reaction was stirred at room temperature for 1.5 hwith exclusion of air and light. After evaporation, the residue wasdissolved in ethyl acetate (100 mL). The mixture was washed withsaturated aqueous NaHCO₃, NaCl, and dried over anhydrous Na₂SO₄. Afterevaporation, the residue was purified by flash column chromatographyusing CH₃OH—CH₂Cl₂ (0˜1:10) as the eluent to afford 21 as yellowcrystals (290 mg; 83% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.31 (s, 1H,6-H), 6.17 (dd, J=6.0, 7.3 Hz, 1H, 1′-H), 5.87-5.97 (m, 1H, CH₂CH═CH₂),5.26-5.33 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.15-5.19 (dm, J=10.4Hz, 1H, one of CH₂CH═CH₂), 4.31 (s, 2H, C≡CCH₂), 4.17 (m, 1H, 3′-H),4.09 (m, 1H, 4′-H), 3.98-4.10 (m, 2H, CH₂CH═CH₂), 3.80 (dd, J=3.4, 12.0Hz, 1H, one of 5′-H), 3.72 (dd, J=3.6, 12.0 Hz, 1H, one of 5′-H),2.46-2.53 (ddd, J=2.9, 5.3, 13.6 Hz, 1H, one of 2′-H), 2.04-2.12 (m, 1H,one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ 166.0, 158.4 (q, J=38 Hz,COCF₃), 156.3, 145.8, 135.6, 117.1 (q, J=284 Hz, COCF₃), 117.0, 91.9,90.7, 88.0, 87.0, 79.8, 75.5, 71.2, 62.8, 39.6, 31.0; HRMS (FAB+) calcdfor C₁₇H₂₀O₅N₄F₃ (M+H⁺): 417.1386, found: 417.1377.

3′-O-Allyl-5-(3-aminoprop-1-ynyl)-2′-deoxycytidine-5′-triphosphate (22).21 (133 mg; 0.319 mmol) and proton sponge (83.6 mg; 0.386 mmol) weredried in a vacuum desiccator over P₂O₅ overnight before dissolving intrimethylphosphate (0.60 mL). Freshly distilled POCl₃ (36 μL; 0.383mmol) was added dropwise at 0° C. and the mixture was stirred for 3 h.Then the solution of tributylammonium pyrophosphate (607 mg) andtributylamine (0.61 mL; 2.56 mmol) in anhydrous DMF (2.56 mL) was wellvortexed and added in one portion at room temperature and the reactionmixture was stirred for 30 min. After that triethylammonium bicarbonatesolution (TEAB) (0.1 M; 16 mL) was added and the mixture was stirred for1 h. Then aqueous ammonia (28%; 16 mL) was added and the reactionmixture was stirred for 12 h. After most liquid was removed undervacuum, the residue was redissolved in water (2 mL) and filtered. Theaqueous solution was purified by DEAE Sephadex A25 ion exchange columnusing gradient aqueous TEAB solution (from 0.1 M to 1.0 M) as eluent toafford 22 as colorless syrup after evaporation: ¹H NMR (300 MHz, D₂O) δ8.43 (s, 1H, 6-H), 6.21 (t, J=6.7 Hz, 1H, 1′-H), 5.85-6.00 (m, 1H,CH₂CH═CH₂), 5.28-5.38 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.19-5.27(dm, J=10.4 Hz, 1H, one of CH₂CH═CH₂), 4.22-4.41 (m, 3H, 3′-H andC≡CCH₂), 4.05-4.18 (m, 3H, 4′-H and CH₂CH═CH₂), 3.94-4.01 (m, 2H, 5′-H),2.47-2.59 (m, 1H, one of 2′-H), 2.20-2.32 (m, 1H, one of 2′-H); ³¹P NMR(121.4 MHz, D₂O) δ −7.1 (d, J=19.8 Hz, 1P, γ-P), −11.1 (d, J=19.1 Hz,1P, α-P), −21.9 (t, J=19.5 Hz, 1P, β-P).

3′-O-Allyl-dCTP-allyl-Bodipy-FL-510 (23). To a stirred solution ofallyl-Bodipy-FL-510 NHS ester 12 in DMF (300 μL), 3′-O-allyl-dCTP-NH₂ 22in 1M NaHCO₃—Na₂CO₃ buffer (300 μL; pH 8.7) was added. The reactionmixture was stirred at room temperature for 7 h and the crude productwas purified by a preparative TLC plate using CH₃OH—CH₂Cl₂ (1:1) as theeluent. The crude product was further purified by reverse-phase HPLCusing a 150×4.6 mm C18 column to afford compound 23 (retention time=35min). Mobile phase: A, 4.4 mM Et₃N/98.3 mM1,1,1,3,3,3-hexafluoro-2-propanol in water (pH=8.1); B, methanol.Elution was performed from 100% A isocratic over 10 min followed by alinear gradient of 0-50% B for 20 min and then 50% B isocratic over 20min. The product was characterized by the following single baseextension reaction to generate DNA extension product 24 andcharacterized by MALDI-TOF MS.

A general procedure of primer extension using3′-O-allyl-dNTPs-allyl-Fluorophore. The polymerase extension reactionmixture consisted of 50 pmol of primer (5′-GTTGATGTACACATTGTCAA-3′, SEQID NO:1), 80 pmol of 100-mer template(5′-TACCCGGAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3′, SEQ ID NO:2), 120pmol of 3′-O-allyl-dNTP-allyl-Fluorophore, 1× Thermopol II reactionbuffer [20 mM Tris-HCl/10 mM (NH₄)₂SO₄/10 mM KCl/2 mM MnCl₂/0.1% TritonX-100, pH 8.8, New England Biolabs], and 6 units of 9° N Polymerase(exo-)A485L/Y409V in a total volume of 20 μL. The reaction consisted of20 cycles at 94° C. for 20 sec, 46° C. for 40 sec, and 60° C. for 90sec. After the reaction, a small portion of the DNA extension productwas desalted using a ZipTip and analyzed by MALDI-TOF MS, which showed adominant peak at m/z 5935 corresponding to the DNA extension productgenerated by incorporating 23. The rest of the product was subjected tothe following deallylation.

General one-pot dual-deallylation procedure of DNA extension products.The DNA product from above (20 pmol) was added to a mixture of degassed1× Thermopol I reaction buffer (20 mM Tris-HCl/10 mM (NH₄)₂SO₄/10 mMKCl/2 mM MgSO₄/0.1% Triton X-100, pH 8.8, 1 μL), Na₂PdCl₄ in degassedH₂O (7 μL, 23 nmol) and P(PhSO₃Na)₃ in degassed H₂O (10 μL, 176 nmol) toperform an one-pot dual-deallylation reaction. The reaction mixture wasthen placed in a heating block and incubated at 70° C. for 30 seconds toyield quantitatively deallylated DNA product and characterized byMALDI-TOF MS as a single peak.

5. Synthesis of 3′-O-allyl-dUTP-allyl-R6G (27)

Synthesis of 3′-O-allyl-dUTP-NH₂ 26 was performed according to theprocedures in reference (28).

3′-O-Allyl-dUTP-allyl-R6G (27). The procedure was similar to thesynthesis of 23. The product was characterized by the single baseextension reaction and MALDI-TOF MS in FIG. 9

6. Synthesis of 3′-O-allyl-dATP-allyl-ROX (39)

6-Chloro-7-iodo-7-deazapurine (31). To a vigorously stirred solution of30 (1.0 g; 6.51 mmol) in CH₂Cl₂ (55 mL), N-iodosuccimide (1.70 g; 7.18mmol) was added. The suspension mixture was stirred at room temperaturefor 1 h, during which more precipitate was observed. The precipitate wasfiltered and then recrystallized in hot methanol to afford 31 (1.49 g;82% yield): ¹H NMR (400 MHz, DMSO-d6) δ 12.96 (br s, 1H, NH), 8.59 (s,1H, 2-H), 7.94 (s, 1H, 8-H); ¹³C NMR (100 MHz, DMSO-d6) δ 151.2, 150.4,150.2, 133.6, 115.5, 51.7; HRMS (FAB+) calcd for C₆H₄N₃ClI (M+H⁺):279.9139, found: 279.9141.

6-Chloro-9-(β-D-2′-deoxyribofuranosyl)-7-iodo-7-deazapurine (33). To astirred solution of 31 (707 mg; 2.53 mmol) in CH₃CN (43 mL), KOH powder(355 mg; 6.34 mmol) and tris [2-(2-methoxyethoxy)ethyl]amine (TDA-1) (52μL, 0.165 mmol) were added. The mixture was stirred at room temperaturefor 10 min and then 3,5-di-O-(p-toluyl)-2-deoxy-D-ribofuranosyl chloride32 (1.18 g; 2.73 mmol) was added. The reaction mixture was stirredvigorously at room temperature for 1 h, and the insoluble portion wasfiltered and washed with hot acetone. The combined solution wasevaporated and dissolved in 7M ammonia in methanol solution (86 mL). Themixture was stirred at room temperature for 24 h. After evaporation, thecrude product was purified by flash column chromatography usingCH₃OH—CH₂Cl₂ (0˜1:20) as the eluent to afford 33 as white solid (711 mg;71% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.57 (s, 1H, 2-H), 8.08 (s, 1H,8-H), 6.72 (dd, J=6.3, 7.5 Hz, 1H, 1′-H), 4.53 (m, 1H, 3′-H), 4.00 (m,1H, 4′-H), 3.80 (dd, J=3.6, 12.0 Hz, 1H, one of 5′-H), 3.74 (dd, J=3.6,12.0 Hz, 1H, one of 5′-H), 2.56-2.64 (ddd, J=6.1, 7.5, 13.5 Hz, 1H, oneof 2′-H), 2.36-2.43 (ddd, J=3.3, 6.2, 13.5 Hz, 1H, one of 2′-H); ¹³C NMR(100 MHz, CD₃OD) δ 152.9, 151.7, 151.3, 134.7, 118.5, 89.0, 85.7, 72.6,63.2, 52.6, 41.7; HRMS (FAB+) calcd for C₁₁H₁₂O₃N₃ClI (M+H⁺): 395.9612,found: 395.9607.

9-[β-D-5′-O-(tert-Butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-iodo7-deazapurine(34).

The procedure was similar to the synthesis of 18, and the crude productwas purified by flash column chromatography using ethyl acetate-hexane(1:3-2) as the eluent to afford 34 as white solid (597 mg; 65% yield)and 33 (213 mg; 30% yield). The above procedure was repeated with therecovered 33 to achieve a 86% overall yield of 34: ¹H NMR (400 MHz,CD₃OD) δ 8.56 (s, 1H, 2-H), 7.99 (s, 1H, 8-H), 6.73 (‘t’, J=6.7 Hz, 1H,1′-H), 4.52 (m, 1H, 3′-H), 4.02 (m, 1H, 4′-H), 3.92 (dd, J=3.0, 11.4 Hz,1H, one of 5′-H), 3.86 (dd, J=3.1, 11.4 Hz, 1H, one of 5′-H), 2.47-2.55(ddd, J=5.8, 7.1, 13.4 Hz, 1H, one of 2′-H), 2.40-2.47 (ddd, J=3.6, 6.3,13.4 Hz, 1H, one of 2′-H), 0.94 (s, 9H, C(CH₃)₃), 0.14 (s, 3H, one ofSiCH₃), 0.13 (s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 152.8,151.5, 151.3, 133.8, 118.2, 88.9, 85.4, 72.5, 64.6, 52.6, 42.4, 26.7,19.5, −4.9, −5.0; HRMS (FAB+) calcd for C₁₇H₂₆O₃N₃ClSiI (M+H⁺):510.0477, found: 510.0487.

9-[β-D-3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-iodo-7-deazapurine(35). To a stirred solution of 34 (789 mg; 1.55 mmol) in CH₂Cl₂ (48 mL),tetrabutylammonium bromide (TBAB) (255 mg; 0.77 mmol), allyl bromide(0.69 mL, 7.74 mmol) and 40% aqueous NaOH solution (24 mL) were addedrespectively. The reaction mixture was stirred at room temperature for 1h. Ethyl acetate (150 mL) was added and the organic layer was separated.The aqueous layer was extracted with ethyl acetate (2×50 mL). Thecombined organic layer was washed with saturated aqueous NaHCO₃, NaCl,and dried over anhydrous Na₂SO₄. After evaporation, the residue waspurified by flash column chromatography using ethyl acetate-hexane (1:6)as the eluent to afford 35 as yellow oil (809 mg; 95% yield): ¹H NMR(400 MHz, CD₃OD) δ 8.52 (s, 1H, 2-H), 7.94 (s, 1H, 8-H), 6.64 (dd,J=6.1, 7.6 Hz, 1H, 1′-H), 5.88-5.99 (m, 1H, CH₂CH═CH₂), 5.28-5.34 (dm,J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.4 Hz, 1H, one ofCH₂CH═CH₂), 4.28 (m, 1H, 3′-H), 4.13 (m, 1H, 4′-H), 4.01-4.11 (m, 2H,CH₂CH═CH₂), 3.88 (dd, J=3.6, 11.2 Hz, 1H, one of 5′-H), 3.80 (dd, J=3.1,11.3 Hz, 1H, one of 5′-H), 2.51-2.57 (ddd, J=2.7, 6.0, 13.5 Hz, 1H, oneof 2′-H), 2.42-2.50 (ddd, J=5.7, 7.7, 13.5 Hz, 1H, one of 2′-H), 0.93(s, 9H, C(CH₃)₃), 0.13 (s, 3H, one of SiCH₃), 0.12 (s, 3H, one ofSiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 152.8, 151.4, 151.3, 135.5, 133.6,118.2, 117.2, 86.5, 85.6, 80.2, 71.0, 64.8, 52.8, 39.7, 26.7, 19.4,−4.8, −5.0; HRMS (FAB+) calcd for C₂₀H₃₀O₃N₃ClSiI (M+H⁺): 550.0790,found: 550.0773.

3′-O-Allyl-7-deaza-7-iodo-2′-deoxyadenosine (36). To a stirred solutionof 35 (809 mg; 1.47 mmol) in anhydrous THF (34 mL), 1 M TBAF in THFsolution (1.62 mL; 1.62 mmol) was added and the reaction was stirred atroom temperature for 1 h. After evaporation, the residue was dissolvedin 7M ammonia in methanol solution (24 mL). The solution was stirred inan autoclave at 115-120° C. for 15 h. After evaporation, the residue waspurified by flash column chromatography using CH₃OH—CH₂Cl₂ (1:20) as theeluent to afford 36 as white solid (514 mg; 84% yield): ¹H NMR (400 MHz,CD₃OD) δ 8.08 (s, 1H, 2-H), 7.56 (s, 1H, 8-H), 6.45 (dd, J=5.8, 8.6 Hz,1H, l′-H), 5.90-6.00 (m, 1H, CH₂CH═CH₂), 5.29-5.35 (dm, J=17.2 Hz, 1H,one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.5 Hz, 1H, one of CH₂CH═CH₂), 4.28(m, 1H, 3′-H), 4.12 (m, 1H, 4′-H), 4.02-4.12 (m, 2H, CH₂CH═CH₂), 3.78(dd, J=3.7, 12.1 Hz, 1H, one of 5′-H), 3.70 (dd, J=3.6, 12.1 Hz, 1H, oneof 5′-H), 2.53-2.61 (ddd, J=5.8, 8.6, 13.6 Hz, 1H, one of 2′-H),2.41-2.47 (ddd, J=2.0, 5.8, 13.5 Hz, 1H, one of 2′-H); ¹³C NMR (100 MHz,CD₃OD) δ 158.5, 152.3, 150.3, 135.7, 128.8, 117.0, 105.3, 86.8, 86.4,80.7, 71.0, 63.7, 51.3, 38.8; HRMS (FAB+) calcd for C₁₄H₁₈O₃N₄I (M+H⁺):417.0424, found: 417.0438.

3′-O-Allyl-7-deaza-7-(3-[(trifluoroacetyl)amino]prop-1-ynyl)-2′-deoxyadenosine(37). The procedure was similar to the synthesis of 21, and the crudeproduct was purified by flash column chromatography using ethylacetate-hexane (1:1˜0) as the eluent to afford 37 as yellow solid (486mg; 90% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.08 (s, 1H, 2-H), 7.60 (s,1H, 8-H), 6.41 (dd, J=5.8, 8.6 Hz, 1H, 1′-H), 5.89-6.00 (m, 1H,CH₂CH═CH₂), 5.29-5.35 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21(dm, J=10.4 Hz, 1H, one of CH₂CH═CH₂), 4.31 (s, 2H, C≡CCH₂), 4.29 (m,1H, 3′-H), 4.13 (m, 1H, 4′-H), 4.01-4.11 (m, 2H, CH₂CH═CH₂), 3.79 (dd,J=3.6, 12.1 Hz, 1H, one of 5′-H), 3.71 (dd, J=3.5, 12.1 Hz, 1H, one of5′-H), 2.54-2.62 (ddd, J=5.8, 8.6, 13.6 Hz, 1H, one of 2′-H), 2.42-2.48(ddd, J=1.9, 5.8, 13.6 Hz, 1H, one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ158.8, 158.6 (q, J=38 Hz, COCF₃), 152.9, 149.6, 135.6, 128.1, 117.1 (q,J=284 Hz, COCF₃), 117.0, 104.5, 96.3, 87.3, 86.9, 86.8, 80.7, 77.0,71.0, 63.8, 38.7, 31.1; HRMS (FAB+) calcd for C₁₉H₂₁O₄N₅F₃ (M+H⁺):440.1546, found: 440.1544.

3′-O-Allyl-7-(3-aminoprop-1-ynyl)-7-deaza-2′-deoxyadenosine-5′-triphosphate(38). The procedure was similar to the synthesis of 22 to yield 38 ascolorless syrup: ¹H NMR (300 MHz, D₂O) δ 8.02 (s, 1H, 2-H), 7.89 (s, 1H,8-H), 6.54 (t, J=6.6 Hz, 1H, 1′-H), 5.89-6.02 (m, 1H, CH₂CH═CH₂),5.30-5.39 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.20-5.27 (dm, J=10.4Hz, 1H, one of CH₂CH═CH₂), 4.48 (s, 2H, C≡CCH₂), 4.35 (m, 1H, 3′-H),4.05-4.17 (m, 4H, CH₂CH═CH₂ and 5′-H), 3.99 (m, 1H, 4′-H), 2.50-2.59 (m,2H, 2′-H); ³¹P NMR (121.4 MHz, D₂O) δ −6.1 (d, J=21.1 Hz, 1P, γ-P),−10.8 (d, J=18.8 Hz, 1P, α-P), −21.9 (t, J=19.9 Hz, 1P, β-P).

3′-O-Allyl-dATP-allyl-ROX (39). The procedure was similar to thesynthesis of 23. The product was characterized by single base extensionreaction and MALDI-TOF MS. See FIG. 10.

7. Synthesis of 3′-O-allyl-dGTP-allyl-Bodipy-650 (43) and3′-O-allyl-dGTP-allyl-Cy5 (44)

Synthesis of 3′-O-allyl-dGTP-NH₂ 42 was performed according to theprocedures in reference (29).

3′-O-Allyl-dGTP-allyl-Bodipy-650 (43) and 3′-O-allyl-dGTP-allyl-Cy5(44). The procedures were similar to the synthesis of 23. The productwas characterized by single base extension reaction and MALDI-TOF MS.See FIG. 11.

II. Synthesis of 3′-O-allyl-dNTPs 1. Synthesis of 3′-O-allyl dCTP (51)

5′-O-(tert-Butyldimethylsilyl)-2′-deoxycytidine (48). To a stirredsolution of 2′-deoxycytidine 47 (1.00 g; 4.40 mmol) in dry pyridine (37mL), TBDMSCl (814 mg; 5.39 mmol) was added. The mixture was stirred atroom temperature for 20 h. After evaporation, the residue was purifiedby flash column chromatography using CH₃OH—CH₂Cl₂ (1:10) as the eluentto afford 48 as white solid (1.26 g; 84% yield): ¹H NMR (400 MHz, CD₃OD)δ 8.03 (d, J=7.5 Hz, 1H, 6-H), 6.23 (t, J=6.3 Hz, 1H, 1′-H), 5.86 (d,J=7.5 Hz, 1H, 5-H), 4.35 (in, 1H, 3′-H), 3.91-3.98 (m, 2H, 4′-H and oneof 5′-H), 3.85 (dd, J=2.5, 11.3 Hz, 1H, one of 5′-H), 2.36-2.43 (ddd,J=4.1, 6.1, 13.5 Hz, 1H, one of 2′-H), 2.05-2.13 (m, 1H, one of 2′-H),0.94 (s, 9H, C(CH₃)₃), 0.14 (s, 3H, one of SiCH₂), 0.13 (s, 3H, one ofSiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 167.1, 157.6, 142.1, 95.6, 88.7,87.4, 71.7, 64.1, 42.7, 26.5, 19.3, −5.2, −5.3; HRMS (FAB+) calcd forC₁₅H₂₈O₄N₃Si (M+H⁺): 342.1849, found: 342.1844.

3′-O-Allyl-5′-O- (tert-butyldimethylsilyl)-2′-deoxycytidine (49). Theprocedure was similar to the synthesis of 19 and the crude product waspurified by flash column chromatography using CH₃OH—CH₂Cl₂ (1:20) as theeluent to afford 49 as yellow solid (480 mg; 35% yield): ¹H NMR (400MHz, CD₃OD) δ 7.97 (d, J=7.5 Hz, 1H, 6-H), 6.21 (t, J=6.5 Hz, 1H, 1′-H),5.87 (d, J=7.5 Hz, 1H, 5-H), 5.87-5.97 (m, 1H, CH₂CH═CH₂), 5.26-5.33(dm, J=17.2 Hz, 1H, one of CH₂CH═CH₂), 5.15-5.20 (dm, J=10.5 Hz, 1H, oneof CH₂CH═CH₂), 4.16 (m, 1H, 3′-H), 4.11 (m, 1H, 4′-H), 3.97-4.11 (m, 2H,CH₂CH═CH₂), 3.92 (dd, J=3.2, 11.4 Hz, 1H, one of 5′-H), 3.84 (dd, J=2.8,11.4 Hz, 1H, one of 5′-H), 2.46-2.51 (ddd, J=3.1, 5.9, 13.6 Hz, 1H, oneof 2′-H), 2.00-2.08 (m, 1H, one of 2′-H), 0.94 (s, 9H, C(CH₃)₃), 0.13(s, 6H, Si(CH₃)₂); ¹³C NMR (100 MHz, CD₃OD) δ 167.2, 157.7, 141.9,135.6, 117.1, 95.7, 87.5, 86.6, 79.7, 71.1, 64.4, 39.8, 26.5, 19.3,−5.1, −5.2; HRMS (FAB+) calcd for C₁₅H₂₈O₄N₃Si (M+H⁺): 342.1849, found:342.1844.

3′-O-Allyl-2′-deoxycytidine (50). The procedure was similar to thesynthesis of 20 and the crude product was purified by flash columnchromatography using CH₃OH-THF (1:12) and CH₃OH-ethyl acetate (1:4) asthe eluent to afford 50 as white foam (269 mg; 80% yield): ¹H NMR (400MHz, CD₃OD) δ 7.96 (d, J=7.5 Hz, 1H, 6-H), 6.21 (dd, J=5.9, 7.6 Hz, 1H,1′-H), 5.89 (d, J=7.5 Hz, 1H, 5-H), 5.87-5.98 (m, 1H, CH₂CH═CH₂),5.27-5.33 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.15-5.19 (dm, J=10.4Hz, 1H, one of CH₂CH═CH₂), 4.17 (m, 1H, 3′-H), 3.98-4.10 (m, 3H, 4′-Hand CH₂CH═CH₂), 3.77 (dd, J=3.6, 12.0 Hz, 1H, one of 5′-H), 3.71 (dd,J=3.7, 12.0 Hz, 1H, one of 5′-H), 2.43-2.50 (ddd, J=2.7, 5.9, 13.6 Hz,1H, one of 2′-H), 2.03-2.11 (ddd, J=6.2, 7.7, 13.6 Hz, 1H, one of 2′-H);¹³C NMR (100 MHz, CD₃OD) δ 167.1, 157.7, 142.2, 135.5, 117.0, 96.0,87.5, 86.6, 80.0, 71.0, 63.0, 39.1; HRMS (FAB+) calcd for C₁₂H₁₈O₄N₃(M+H⁺): 268.1297, found: 268.1307.

3′-O-Allyl-2′-deoxycytidine-5′-triphosphate (51). 50 (86 mg; 0.32 mmol)and proton sponge (84 mg; 0.39 mmol) were dried in a vacuum desiccatorover P₂O₅ overnight before dissolving in trimethylphosphate (0.60 mL).

Freshly distilled POCl₃ (35.8 μL; 0.383 mmol) was added dropwise at 0°C. and the mixture was stirred for 3 h. Then the solution oftributylammonium pyrophosphate (607 mg) and tributylamine (0.61 mL; 2.56mmol) in anhydrous DMF (2.6 mL) was well vortexed and added in oneportion at room temperature and the reaction was stirred for 30 min.After that triethylammonium bicarbonate solution (TEAB) (0.1 M; 16 mL)was added and the mixture was stirred for 2 h. After most liquid wasremoved under vacuum, the residue was redissolved in water (2 mL) andfiltered. The aqueous solution was purified by DEAE Sephadex A25 ionexchange column using gradient aqueous TEAB solution (from 0.1 M to 1.0M) as eluent to afford 51 as colorless syrup after evaporation: ¹H NMR(300 MHz, D₂O) δ 7.90 (d, J=7.4 Hz, 1H, 6-H), 6.20 (dd, J=5.9, 7.6 Hz,1H, 1′-H), 5.92 (d, J=7.4 Hz, 1H, 5-H), 5.85-5.97 (m, 1H, CH₂CH═CH₂),5.25-5.34 (m, 1H, one of CH₂CH═CH₂), 5.15-5.20 (m, 1H, one ofCH₂CH═CH₂), 4.15 (m, 1H, 3′-H), 3.96-4.10 (m, 3H, 4′-H and CH₂CH═CH₂),3.70-3.80 (m, 2H, 5′-H), 2.43-2.52 (m, 1H, one of 2′-H), 2.05-2.14 (m,1H, one of 2′-H); ³¹P NMR (121.4 MHz, D₂O) δ −8.8 (d, J=19.0 Hz, 1P,γ-P), −11.3 (d, J=19.6 Hz, 1P, α-P), −22.9 (t, J=19.5 Hz, 1P, β-P).

2. Synthesis of 3′-O-allyl-dTTP (53)

Synthesis of 3′-O-allylthymidine 52 was performed according to theprocedures in reference (28).

3′-O-Allylthymidine-5′-triphosphate (53). The procedure was similar tothe synthesis of 51 to yield 53 as colorless syrup: ¹H NMR (300 MHz,D₂O) δ 7.80 (m, 1H, 6-H), 6.23 (dd, J=6.2, 8.1 Hz, 1H, 1′-H), 5.85-5.97(m, 1H, CH₂CH═CH₂), 5.25-5.32 (m, 1H, one of CH₂CH═CH₂), 5.15-5.21 (m,1H, one of CH₂CH═CH₂), 4.17 (m, 1H, 3′-H), 3.97-4.11 (m, 3H, 4′-H andCH₂CH═CH₂), 3.70-3.80 (m, 2H, 5′-H), 2.30-2.41 (m, 1H, one of 2′-H),2.11-2.23 (m, 1H, one of 2′-H), 1.86 (d, J=1.2 Hz, 3H, CH₃); ³¹P NMR(121.4 MHz, D₂O) δ −7.1 (d, J=20.1 Hz, 1P, γ-P), −10.8 (d, J=19.5 Hz,1P, α-P), −21.8 (t, J=19.5 Hz, 1P, β-P).

3. Synthesis of 3′-O-allyl-dATP (59)

N⁶-[(Dimethylamino)methylene]-2′-deoxyadenosine (55).

To a stirred solution of 2′-deoxyadenosine monohydrate 54 (1.00 g; 3.71mmol) in methanol (43 mL), N,N-dimethylformamide dimethyl acetal (2.48mL; 18.6 mmol) was added. The reaction was stirred at 50° C. for 16 h.After evaporation, CH₂Cl₂-hexane (1:1) was added. The white solid formedwas then filtered, collected and washed by hexane to afford 55 as whitesolid (1.13 g; 99% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.92 (s, 1H,CHN(CH₃)₂), 8.44 (s, 1H, 2-H), 8.43 (s, 1H, 8-H), 6.48 (dd, J=6.2, 7.8Hz, 1H, 1′-H), 4.59 (m, 1H, 3′-H), 4.07 (m, 1H, 4′-H), 3.86 (dd, J=3.1,12.2 Hz, 1H, one of 5′-H), 3.76 (dd, J=3.5, 12.2 Hz, 1H, one of 5′-H),3.25 (s, 3H, one of NCH₃), 3.24 (s, 3H, one of NCH₃), 2.80-2.88 (ddd,J=5.9, 7.8, 13.5 Hz, 1H, one of 2′-H), 2.40-2.47 (ddd, J=2.8, 6.1, 13.4Hz, 1H, one of 2′-H); =³C NMR (100 MHz, CD₃OD) δ 161.0, 159.9, 152.8,151.1, 142.8, 127.0, 89.7, 86.9, 72.9, 63.5, 41.5 (N(CH₃)₂), 35.3; HRMS(FAB+) calcd for C₁₃H₁₉O₃N₆ (M+H⁺): 307.1519, found: 307.1511.

5′-O-(tert-Butyldimethylsilyl)-N⁶-[(dimethylamino)methylene]-2′-deoxyadenosine(56). The procedure was similar to the synthesis of 18, and the crudeproduct was purified by flash column chromatography using CH₃OH—CH₂Cl₂(1:20) as the eluent to afford 56 as white foam (1.11 g; 72% yield): ¹HNMR (400 MHz, CD₃OD) δ 8.90 (s, 1H, CHN(CH₃)₂), 8.45 (s, 1H, 2-H), 8.43(s, 1H, 8-H), 6.49 (t, J=6.5 Hz, 1H, 1′-H), 4.59 (m, 1H, 3′-H), 4.04 (m,1H, 4′-H), 3.95 (dd, J=3.7, 11.3 Hz, 1H, one of 5′-H), 3.85 (dd, J=2.8,11.3 Hz, 1H, one of 5′-H), 3.25 (s, 3H, one of NCH₃), 3.24 (s, 3H, oneof NCH₃), 2.73-2.81 (m, 1H, one of 2′-H), 2.48-2.55 (ddd, J=4.0, 6.4,13.5 Hz, 1H, one of 2′-H), 0.90 (s, 9H, C(CH₃)₃), 0.08 (s, 3H, one ofSiCH₃), 0.07 (s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 160.6,159.7, 153.0, 151.7, 141.8, 126.5, 88.9, 85.7, 72.1, 64.3, 41.6, 41.5,35.3, 26.5, 19.3, −5.0, −5.1; HRMS (FAB+) calcd for C₁₉H₃₃O₃N₆Si (M+H⁺):421.2383, found: 421.2390.

3′-O-Allyl-5′-O-(t-butyldimethylsilyl)-N⁶-[(dimethylamino)methylene]-2′-deoxyadenosine(57). The procedure was similar to the synthesis of 35, and the crudeproduct was purified by flash column chromatography using CH₃OH—CH₂Cl₂(1:25) and CH₃OH-ethyl acetate (1:10) as the eluent to afford 57 ascolorless oil (875 mg; 72% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.90 (s,1H, CHN(CH₃)₂), 8.44 (s, 1H, 2-H), 8.41 (s, 1H, 8-H), 6.45 (dd, J=6.3,7.2 Hz, 1H, 1′-H), 5.91-6.01 (m, 1H, CH₂CH═CH₂), 5.30-5.37 (dm, J=17.2Hz, 1H, one of CH₂CH═CH₂), 5.18-5.22 (dm, J=10.5 Hz, 1H, one ofCH₂CH═CH₂), 4.37 (m, 1H, 3′-H), 4.17 (m, 1H, 4′-H), 4.05-4.15 (m, 2H,CH₂CH═CH₂), 3.91 (dd, J=4.6, 11.1 Hz, 1H, one of 5′-H), 3.83 (dd, J=3.8,11.1 Hz, 1H, one of 5′-H), 3.25 (s, 3H, one of NCH₃), 3.24 (s, 3H, oneof NCH₃), 2.76-2.83 (ddd, J=6.0, 7.3, 13.6 Hz, 1H, one of 2′-H),2.59-2.65 (ddd, J=3.0, 6.1, 13.6 Hz, 1H, one of 2′-H), 0.90 (s, 9H,C(CH₃)₃), 0.08 (s, 6H, Si(CH₃)₂); ¹³C NMR (100 MHz, CD₃OD) δ 160.7,159.7, 153.1, 151.8, 141.9, 135.6, 126.5, 117.1, 86.7, 85.9, 80.1, 71.1,64.5, 41.5, 38.7, 35.3, 26.5, 19.3, −5.0, −5.1; HRMS (FAB+) calcd forC₂₂H₃₇O₃N₆Si (M+H⁺): 461.2696, found: 461.2695.

3′-O-Allyl-2′-deoxyadenosine (58). To a stirred solution of 57 (875 mg;1.90 mmol) in anhydrous THF (45 mL), 1 M TBAF in THF solution (2.09 mL;2.09 mmol) was added and the reaction was stirred at room temperaturefor 1 h. After evaporation, the residue was dissolved in 7 M ammonia inmethanol solution (34 mL). The mixture was then stirred in a sealedflask at 50° C. for 9 h. After evaporation, the residue was purified byflash column chromatography using CH₃OH—CH₂Cl₂ (1:10) as the eluent toafford 58 as white solid (548 mg; 99% yield): ¹H NMR (400 MHz, CD₃OD) δ8.30 (s, 1H, 2-H), 8.17 (s, 1H, 8-H), 6.38 (dd, J=5.8, 8.6 Hz, 1H,1′-H), 5.91-6.01 (m, 1H, CH₂CH═CH₂), 5.30-5.37 (dm, J=17.3 Hz, 1H, oneof CH₂CH═CH₂), 5.17-5.22 (dm, J=10.6 Hz, 1H, one of CH₂CH═CH₂), 4.36 (m,1H, 3′-H), 4.21 (m, 1H, 4′-H), 4.04-4.15 (m, 2H, CH₂CH═CH₂), 3.85 (dd,J=3.2, 12.3 Hz, 1H, one of 5′-H), 3.74 (dd, J=3.2, 12.3 Hz, 1H, one of5′-H), 2.75-2.83 (ddd, J=5.7, 8.6, 13.6 Hz, 1H, one of 2′-H), 2.52-2.58(ddd, J=1.8, 5.8, 13.6 Hz, 1H, one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ157.1, 153.1, 149.5, 141.2, 135.6, 120.6, 117.0, 87.5, 87.2, 80.9, 71.0,63.9, 38.7; HRMS (FAB+) calcd for C₁₃H₁₈O₃N₅ (M+H⁺): 292.1410, found:292.1426.

3′-O-Allyl-2′-deoxyadenosine-5′-triphosphate (59). The procedure wassimilar to the synthesis of 51 to yield 59 as colorless syrup: ¹H NMR(300 MHz, D₂O) δ 8.46 (s, 1H, 2-H), 8.19 (s, 1H, 8-H), 6.43 (dd, J=6.3,7.2 Hz, 1H, 1′-H), 5.90-6.02 (m, 1H, CH₂CH═CH₂), 5.31-5.40 (dm, J=17.1Hz, 1H, one of CH₂CH═CH₂), 5.21-5.28 (dm, J=10.8 Hz, 1H, one ofCH₂CH═CH₂), 4.55 (m, 1H, 3′-H), 4.40 (m, 1H, 4′-H), 4.06-4.20 (m, 4H,CH₂CH═CH₂ and 5′-H), 2.61-2.82 (m, 2H, 2′-H); ³¹P NMR (121.4 MHz, D₂O) δ−8.9 (d, J=19.1 Hz, 1P, γ-P), −11.2 (d, J=19.7 Hz, 1P, α-P), −22.8 (t,J=19.9 Hz, 1P, β-P).

4. Synthesis of 3′-O-allyl-dGTP (65)

2-Amino-6-chloro-9-[β-D-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]purine (61). The procedure was similar to the synthesis of 18, and thecrude product was purified by flash column chromatography usingCH₃OH—CH₂Cl₂ (1:20) as the eluent to afford 61 as white solid (1.20 g;86% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.25 (s, 1H, 8-H), 6.34 (t, J=6.4Hz, 1H, 1′-H), 4.56 (m, 1H, 3′-H), 4.01 (m, 1H, 4′-H), 3.90 (dd, J=3.5,11.4 Hz, 1H, one of 5′-H), 3.84 (dd, J=3.8, 11.4 Hz, 1H, one of 5′-H),2.67-2.74 (m, 1H, one of 2′-H), 2.43-2.50 (ddd, J=4.2, 6.4, 13.5 Hz, 1H,one of 2′-H), 0.89 (s, 9H, C(CH₃)₃), 0.07 (s, 3H, one of SiCH₃), 0.06(s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 161.1, 154.2, 151.2,142.0, 124.9, 88.9, 85.5, 72.0, 64.3, 41.4, 26.5, 19.3, −5.1 (two SiCH);HRMS (FAB+) calcd for C₁₆H₂₇O₃N₅ClSi (M+H⁺): 400.1572, found: 400.1561.

2-Amino-6-chloro-9-[β-D-3′-O-allyl-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribo-furanosyl]-purine(62). The procedure was the same as that of 35, and the crude product 61converted from 60 was purified by flash column chromatography usingethyl acetate-hexane (1:2) as the eluent to afford 62 as white solid(832 mg; 63% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.23 (s, 1H, 8-H), 6.30(t, J=6.7 Hz, 1H, 1′-H), 5.89-5.99 (m, 1H, CH₂CH═CH₂), 5.28-5.35 (dm,J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.5 Hz, 1H, one ofCH₂CH═CH₂), 4.33 (m, 1H, 3′-H), 4.13 (m, 1H, 4′-H), 4.03-4.12 (m, 2H,CH₂CH═CH₂), 3.86 (dd, J=4.3, 11.2 Hz, 1H, one of 5′-H), 3.81 (dd, J=3.9,11.2 Hz, 1H, one of 5′-H), 2.68-2.75 (m, 1H, one of 2′-H), 2.53-2.59(ddd, J=3.2, 6.2, 13.6 Hz, 1H, one of 2′-H), 0.88 (s, 9H, C(CH₃)₃), 0.08(s, 3H, one of SiCH₃), 0.07 (s, 3H, one of SiCH₃); ¹³C NMR (100 MHz,CD₃OD) δ 161.1, 154.2, 151.2, 141.9, 135.5, 124.9, 117.1, 86.7, 85.6,80.0, 71.1, 64.5, 38.7, 26.5, 19.3, −5.1, −5.2; HRMS (FAB+) calcd forC₁₉H₃₁O₃N₅ClSi (M+H⁺): 440.1885, found: 440.1870.

3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (63). To astirred suspension of 95% NaH power (223 mg; 8.83 mmol) in anhydrous THF(82 mL), 3-hydroxypropionitrile (550 μL; 8.00 mmol) was added and themixture was stirred at room temperature for 20 min. Then 62 (832 mg;1.89 mmol) in anhydrous THF (20 mL) was added and the mixture wasstirred at 40° C. for 1 h. At room temperature, 80% acetic acid (630 μL;8.83 mmol) was added and stirred for 20 min. After evaporation, ethylacetate (100 mL) was added. The mixture was washed by saturated aqueousNaHCO₃, NaCl, and dried over anhydrous Na₂SO₄. After evaporation, theresidue was purified by flash column chromatography using CH₃OH—CH₂Cl₂(1:20) as the eluent to afford 63 as white solid (661 mg; 83% yield): ¹HNMR (400 MHz, CD₃OD) δ 7.92 (s, 1H, 8-H), 6.22 (dd, J=6.4, 7.3 Hz, 1H,1′-H), 5.89-5.99 (m, 1H, CH₂CH═CH₂), 5.29-5.35 (dm, J=17.3 Hz, 1H, oneof CH₂CH═CH₂), 5.17-5.21 (dm, J=10.5 Hz, 1H, one of CH₂CH═CH₂), 4.30 (m,1H, 3′-H), 4.11 (m, 1H, 4′-H), 4.03-4.12 (m, 2H, CH₂CH═CH₂), 3.79-3.86(m, 2H, 5′-H), 2.56-2.64 (ddd, J=5.9, 7.4, 13.5 Hz, 1H, one of 2′-H),2.49-2.55 (ddd, J=3.0, 6.1, 13.5 Hz, 1H, one of 2′-H), 0.91 (s, 9H,C(CH₃)₃), 0.10 (s, 3H, one of SiCH₃), 0.09 (s, 3H, one of SiCH₃); ¹³CNMR (100 MHz, CDCl₃) δ 158.7, 153.4, 151.0, 135.3, 134.1, 117.2, 117.1,85.0, 83.8, 78.7, 70.2, 63.3, 38.2, 26.1, 18.5, −5.1, −5.3; HRMS (FAB+)calcd for C₁₉H₃₂O₄N₅Si (M+H⁺): 422.2224, found: 422.2209.

3′-O-Allyl-2′-deoxyguanosine (64). The procedure was similar to thesynthesis of 20 and the crude product was purified by flash columnchromatography using CH₃OH—CH₂Cl₂ (1:10) as the eluent to afford 64 aswhite solid (434 mg; 90% yield): ¹H NMR (400 MHz, CD₃OD) δ 7.94 (s, 1H,8-H), 6.22 (dd, J=5.9, 8.4 Hz, 1H, 1′-H), 5.90-6.00 (m, 1H, CH₂CH═CH₂),5.29-5.36 (dm, J=17.2 Hz, 1H, one of CH₂CH═CH₂), 5.17-5.21 (dm, J=10.5Hz, 1H, one of CH₂CH═CH₂), 4.31 (m, 1H, 3′-H), 4.14 (m, 1H, 4′-H),4.03-4.13 (m, 2H, CH₂CH═CH₂), 3.80 (dd, J=3.8, 12.0 Hz, 1H, one of5′-H), 3.72 (dd, J=3.7, 12.0 Hz, 1H, one of 5′-H), 2.63-2.71 (ddd,J=5.9, 8.4, 13.6 Hz, 1H, one of 2′-H), 2.45-2.52 (ddd, J=2.1, 5.9, 13.6Hz, 1H, one of 2′-H); ¹³C NMR (100 MHz, DMSO-d6) δ 156.4, 153.4, 150.6,135.1, 134.8, 116.5, 116.3, 84.9, 82.6, 79.1, 69.0, 61.8, 36.4; HRMS(FAB+) calcd for C₁₃H₁₈O₄N₅ (M+H⁺): 308.1359, found: 308.1358.

3′-O-Allyl-2′-deoxyguanosine-5′-triphosphate (65). The procedure wassimilar to the synthesis of 51 to yield 65 as colorless syrup: ¹H NMR(300 MHz, D₂O) δ 7.90 (s, 1H, 8-H), 6.21 (dd, J=6.1, 8.1 Hz, 1H, 1′-H),5.86-5.96 (m, 1H, CH₂CH═CH₂), 5.27-5.35 (m, 1H, one of CH₂CH═CH₂),5.15-5.20 (m, 1H, one of CH₂CH═CH₂), 4.30 (m, 1H, 3′-H), 4.15 (m, 1H,4′-H), 4.02-4.14 (m, 2H, CH₂CH═CH₂), 3.75-3.85 (m, 2H, 5′-H), 2.60-2.73(m, 1H, one of 2′-H), 2.42-2.50 (m, 1H, one of 2′-H); ³¹P NMR (121.4MHz, D₂O) δ −10.9 (d, J=18.9 Hz, 1P, γ-P), −11.3 (d, J=19.6 Hz, 1P,α-P), −22.9 (t, J=19.6 Hz, 1P, β-P).

III. Construction of a Chip with Immobilized Self-priming DNA Template.

The DNA chip was constructed as shown in FIG. 5 and involved thefollowing three steps:

Synthesis of the alkyne-functionalized DNA template. The5′-amino-hairpin DNA templates (GeneLink, N.Y.)5′-NH₂-TTT-TTG-TTT-TTT-TTT-TCG-ATC-GAC-TTA-AGG-CGC-TTG-CGC-CTT-AAG-TCG-3′(SEQ ID NO:3) and5′-NH₂-AGT-CAG-TCT-CTC-ATC-TCG-ACA-TCT-ACG-CTA-CTC-GTC-GAT-CGG-AAA-CAG-CTA-TGA-CCA-TGC-TTG-CAT-GGT-CAT-AGC-TGT-TTC-C-3′(SEQ ID NO:4) were coupled with 6-heptynoic acid by adding 300 μL DMSOsolution of 6-heptynoic-NHS ester [succinimidyl N-(6-heptynoate)] (0.8M) into the 1000 μL DNA template solution (200 μM, in 0.25 MNa₂CO₃/NaHCO₃ buffer, pH 9.0). The reaction mixture was stirred for 5 hat room temperature to produce a terminal alkynyl group on the 5′-end ofthe hairpin DNA. The resulting alkyne-functionalized DNA was separatedfrom the excess reagent by size-exclusion chromatography using PD-10columns (GE Health, N.J.). Further desalting with an oligonucleotidepurification cartridge (Applied Biosystems, Calif.) and drying affordedthe crude product which was further purified by reverse-phase HPLC(Waters HPLC system containing Waters Delta 600 controller, Rheodyne77251 injector and 2996 photodiode array detector, Milford, Mass.) usinga C-18 reverse column (Xterra MS C18, 4.6 mm×50 mm, 2.5 μm) at a flowrate of 0.5 mL/min, with detection at 260 nm, and elution with a lineargradient of 12-34.5% of B in A over 40 min (A: 4.4 mM triethylamine and100 mM hexafluoroisopropyl alcohol aqueous solution, pH 8.1; B:methanol). The fractions containing the desired product were collectedand evaporated to dryness under vacuum. MALDI-TOF MS was used tocharacterize the product on a Voyager DE matrix assisted laserspectrometer (Applied Biosystems, Calif.) using 3-hydroxypicolinic acidas a matrix.

Azide functionalization of an amine-modified glass surface. Theamine-modified glass slide (Corning® GAPS II) was cleaned andpre-treated by immersion into a basic solution [N,N-diisopropylethylamine (DIPEA)/dimethylformamide (DMF), 1:9 (V/V)] for 30 min. Theglass slide was then washed with DMF, and transferred into the 2 mL DMFcoupling solution containing 100 mMO-(2-azidoethyl)-O′-[2-(diglycolyl-amino)-ethyl]heptaethylene glycol(Fluka, Switzerland),Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) (Novabiochem, Calif.) and 200 mM DIPEA. The reaction vessel wasgently shaken for 4 h at room temperature. The azide functionalizedglass slide was washed thoroughly with DMF and ethanol, and then driedunder argon gas stream.

DNA immobilization on the azide-modified glass surface using 1,3-dipolaralkyne-azide cycloaddition chemistry.

A coupling mixture was prepared by mixing tetrakis-(acetonitrile)copper(I) hexafluorophosphate (2 mM/DMSO), tris-(benzyltriazolylmethyl) amine(TBTA) (2 mM/DMSO), sodium ascorbate (2.6 mM/H₂O) and alkynyl DNA (50μM/H₂O) with a volumetric ratio of 3:3:2.3:3. This coupling mixture wasthen spotted onto the azide-functionalized glass slide in the form of11.0 μL drops with the aid of adhesive silicone isolators (GraceBio-Labs, Oreg.) to create uniform spots on the glass surface. The DNAspotted glass slide was incubated in a humid chamber at room temperaturefor 8 h, then washed with de-ionized water and SPSC buffer (50 mM sodiumphosphate/l M NaCl, pH 7.5) for ½ h to remove non-specifically boundDNA, and finally rinsed with dH₂O. The formation of a stable hairpin wasascertained by covering the DNA spots with 1× Thermolpol II reactionbuffer (10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl, 0.1% Triton X-100, 4mM MnCl₂, pH 8.8), incubating it in a humid chamber at 95° C. for 5 minto dissociate any partial hairpin structure, and then cooling slowly forre-annealing.

IV. Continuous DNA polymerase reaction using four chemically cleavablefluorescent nucleotides as reversible terminators in solution.

We characterized the four nucleotide analogues3′-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′-O-allyl-dUTP-allyl-R6G,3′-O-allyl-dATP-allyl-ROX and 3′-O-allyl-dGTP-allyl-Bodipy-650, byperforming four continuous DNA-extension reactions sequentially using aprimer (5′-AGAGGATCCAACCGAGAC-3′, SEQ ID NO:5) and a synthetic DNAtemplate (5′-GTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGG-3′, SEQ ID NO:6) based on a portion of exon 7 of the human p53gene. The four nucleotides in the template immediately adjacent to theannealing site of the primer are 3′-ACTG-5′. First, a polymeraseextension reaction using a pool of all four nucleotide analogues alongwith the primer and the template was performed producing a single baseextension product. The reaction mixture for this, and all subsequentextension reactions, consisted of 80 pmol of template, 50 pmol ofprimer, 100 pmol of 3′-O-allyl-dNTPs-allyl-fluorophore, 1× Thermopol IIreaction buffer, 40 nmol of Mn²⁺ and 2 U of 9°N mutant DNA polymerase(exo-) A485L/Y409V in a total volume of 20 μL. The reaction consisted of20 cycles at 94° C. for 20 sec, 48° C. for 40 sec, and 62° C. for 90sec. Subsequently, the extension product was purified by usingreverse-phase HPLC. The fraction containing the desired DNA product wascollected and freeze-dried for analysis using MALDI-TOF massspectrometry. For deallylation, the purified DNA extension productbearing the fluorescent nucleotide analogue was resuspended in degassedwater and added to a deallylation cocktail [1× Thermopol I reactionbuffer/Na₂PdCl₄/P(PhSO₃Na)₃] and incubated for 30 s to yield deallylatedDNA product which was characterized by MALDI-TOF MS. The DNA productwith both the fluorophore and the 3′-O-allyl group removed to generate afree 3′-OH group was used as a primer for a second extension reactionusing 3′-O-allyl-dNTPs-allyl-fluorophore. The second extended DNAproduct was then purified by HPLC and deallylated. The third and thefourth extensions were carried out in a similar manner using thepreviously extended and deallylated product as the primer.

V. 4-Color SBS reaction on a chip with four chemically cleavablefluorescent nucleotides as reversible terminators.

Ten microliters of a solution consisting of3′-O-allyl-dCTP-allyl-Bodipy-FL-510 (3 pmol), 3′-O-allyl-dUTP-allyl-R6G(10 pmol), 3′-O-allyl-dATP-allyl-ROX (5 pmol) and3′-O-allyl-dGTP-allyl-Cy5 (2 pmol), 1 U of 9°N mutant DNA polymerase,and 1× Thermolpol II reaction buffer was spotted on the surface of thechip, where the self-primed DNA moiety was immobilized. The nucleotideanalogue complementary to the DNA template was allowed to incorporateinto the primer at 68° C. for 10 min. To synchronize any unincorporatedtemplates, an extension solution consisting of 30 pmol each of3′-O-allyl-dCTP, 3′-O-allyl-dTTP, 3′-O-allyl-dATP and 3′-O-allyl-dGTP, 1U of 9°N mutant DNA polymerase, and 1× Thermolpol II reaction buffer wasspotted on the same spot and incubated at 68° C. for 10 min. Afterwashing the chip with a SPSC buffer containing 0.1% Tween 20 for 5 min,the surface was rinsed with dH₂O, dried briefly and then scanned with a4-color ScanArray Express scanner (Perkin-Elmer Life Sciences) to detectthe fluorescence signal. The 4-color scanner is equipped with fourlasers with excitation wavelengths of 488, 543, 594, and 633 nm andemission filters centered at 522, 570, 614, and 670 nm. Fordeallylation, the chip was immersed in a deallylation cocktail [1×Thermolpol I reaction buffer/Na₂PdCl₄/P(PhSO₃Na)₃] and incubated for 5min at 60° C. The chip was then immediately immersed in a 3 M Tris-HClbuffer (pH 8.5) and incubated for 5 min at 60° C. Finally, the chip wasrinsed with acetonitrile/dH₂O (1:1, V/V) and dH₂O. The chip surface wasscanned again to compare the intensity of fluorescence afterdeallylation with the original fluorescence intensity. This process wasfollowed by the next polymerase extension reaction using3′-O-allyl-dNTPs-allyl-fluorophore and 3′-O-allyl-dNTPs, with thesubsequent washing, fluorescence detection, and deallylation processesperformed as described above. The same cycle was repeated multiple timesusing the four chemically cleavable fluorescent nucleotide mixture inpolymerase extension reaction to obtain de novo DNA sequencing data onvarious different DNA templates.

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1-33. (canceled)
 34. A plurality of different deoxyribonucleic acidscovalently immobilized on a solid support, wherein at least one of theplurality of different deoxyribonucleic acids comprises apolymerase-incorporated labeled nucleotide analogue derived from thegroup consisting of labeled nucleotide analogues A-D, and wherein atleast one of the plurality of different deoxyribonucleic acids comprisesa polymerase-incorporated un-labeled nucleotide analogue derived fromthe group consisting of un-labeled nucleotide analogues A′-D′

wherein R (a) represents a cleavable, chemical group capping the oxygenat the 3′ position of the deoxyribose of the deoxyribonucleotideanalogue, (b) does not interfere with recognition of the analogue as asubstrate by the DNA polymerase or with incorporation of the analogueinto the growing DNA strand during the DNA polymerase reaction, and (c)is stable during the DNA polymerase reaction; wherein the covalent bondbetween the 3′-oxygen and R is stable during the DNA polymerasereaction; wherein each of Dye 1, Dye 2, Dye 3 and Dye 4 represents adistinguishable, detectable moiety; wherein (i) the nucleotide analogueis incorporated into the growing DNA strand as a result of the DNApolymerase reaction, (ii) the incorporated analogue is identified, and(iii) the covalent bond between the 3′-oxygen and R is cleaved underconditions compatible with DNA sequencing to allow the incorporation anddetection of the next nucleotide analogue; wherein Y represents acleavable, chemical linker which (a) does not interfere with recognitionof the analogue as a substrate by the DNA polymerase or withincorporation of the analogue into the growing DNA strand during the DNApolymerase reaction and (b) is stable during the DNA polymerasereaction; wherein the nucleotide analogue: i) is recognized as asubstrate by the DNA polymerase for incorporation into the growing DNAstrand during the DNA polymerase reaction, ii) is efficiently andaccurately incorporated at the end of the growing DNA strand during theDNA polymerase reaction, iii) produces a 3′-OH group on the deoxyriboseupon cleavage of R under conditions compatible with DNA sequencing, andiv) no longer includes a detectable moiety on the base upon cleavage ofY under conditions compatible with DNA sequencing; and wherein if thenucleotide analogue is: (A) or (A′), it forms hydrogen bonds withcytosine or a cytosine nucleotide analogue; (B) or (B′), it formshydrogen bonds with thymine or a thymine nucleotide analogue; (C) or(C′), it forms hydrogen bonds with guanine or a guanine nucleotideanalogue; or (D) or (D′), it forms hydrogen bonds with adenine or anadenine nucleotide analogue.
 35. The plurality of differentdeoxyribonucleic acids of claim 34, wherein the polymerase-incorporatedlabeled nucleotide analogue is derived from A and thepolymerase-incorporated un-labeled nucleotide analogue is derived fromA′.
 36. The plurality of different deoxyribonucleic acids of claim 34,wherein the polymerase-incorporated labeled nucleotide analogue isderived from B and the polymerase-incorporated un-labeled nucleotideanalogue is derived from B′.
 37. The plurality of differentdeoxyribonucleic acids of claim 34, wherein the polymerase-incorporatedlabeled nucleotide analogue is derived from C and thepolymerase-incorporated un-labeled nucleotide analogue is derived fromC′.
 38. The plurality of different deoxyribonucleic acids of claim 34,wherein the polymerase-incorporated labeled nucleotide analogue isderived from D and the polymerase-incorporated un-labeled nucleotideanalogue is derived from D′.